JP2021101720A - Anti-coagulation factor xi antibody - Google Patents

Abstract

To provide a novel therapy having a better safety profile in preventing and treating a thrombotic disease or disorder.SOLUTION: An antibody that binds to an apple 3 domain of a human coagulation factor XI and inhibits activation of FXI by a coagulation factor XIIa and activation of FIX by FXIa.SELECTED DRAWING: Figure 1A

Description

Cross-reference to related applications This application is filed on June 14, 2016, US Provisional Patent Application No. 62 / 349,888 (
It claims the benefits of (which is incorporated herein by reference in its entirety).

(1) Field of Invention The present invention is an antibody that binds to the apple 3 domain of human coagulation factor XI (FXI) and inhibits the activation of FXI by the coagulation factor XIIa and the activity of FXIa against factor IX (FIX). Regarding.

(2) Description of Related Techniques For thromboembolic disorders, including both venous and arterial thrombosis, numerous classes of anticoagulants such as vitamin K antagonists (VKA), heparin and direct thrombin inhibitors are available. Despite this, it remains a major cause of morbidity and mortality in Western countries (Weitz et al., Chest 2008, 133: 234S-256S; H.
awkins, Pharmacotherapy 2004, 24: 62S-65S).
Although these drugs are effective in reducing the risk of thrombosis, they carry a number of restrictions. For example, VKA (eg, warfarin) is the mainstream of oral anticoagulant therapy, but the management of VKA therapy is complex due to its significant bleeding risk, slow onset and offset of action, and numerous interactions between diet and drugs. Yes (Hawkins, supra; Ansell J et al.,
Chest 2008, 133: 160S-198S). Non-vitamin K antagonist Oral anticoagulants including N baroxaban, apixaban, edoxaban and dabigatran
OAC) is at least as effective as warfarin, has less food-drug interactions, and requires no monitoring. However, NOAC still increases the risk of bleeding, as evidenced by the close annual incidence of major or non-major clinical-related bleeding in its enrolled trials for stroke prevention in atrial fibrillation (Con).
noly et al., N Engl J Med 2009, 361: 1139-1151; P
atel et al., N Engl J Med 2011, 365: 883-891; Gran
ger et al., N Engl J Med 2011, 365: 981-992; Giugl
iano et al., N Engl J Med 2013, 369: 2093-2104). This is because NOAC is an essential protein for normal coagulation (hemostatic) (coagulation factor Xa (FXa)).
) And thrombin) are mainly due to the fact that they are targeted. Therefore, there is still a need for new therapies with a better safety profile in the prevention and treatment of thrombotic diseases or disorders.

In the classical waterfall model of the blood coagulation cascade (FIG. 1A), coagulation is initiated by either the extrinsic (tissue factor (TF) activation) pathway or the endogenous (contact activation) pathway, both of which are Enters a common pathway that ultimately results in thrombin production and fibrin formation (Furie & Furie, Cell 1988, 53: 505-51)
8; Galiani & Rennes, J Thromb Haemost 2007,
5: 1106-1112). Extrinsic cascade is initiated when TF present in the subepithelial and atherosclerotic lesions is exposed to flowing blood and forms a complex with the coagulation factor VIIa (FVIIa). The TF-FVIIa complex (exogenous tenase complex) then initiates a common pathway, ie, activates FX to form FXa, which in turn converts prothrombin to thrombin. In addition, the TF-FVIIa complex can activate the coagulation factor IX (FIX) to form Factor IX. FIXa, which forms a complex (endogenous tenase complex) with coagulation factor VIII (FVIIIa), can also cleave the FX substrate. The endogenous cascade is F by contact activation from the charged surface (eg, collagen and glycosaminoglycans).
It is initiated when XIIa is formed and propagates thrombin production by sequential activation of FXI, FIX, FX and prothrombin. Thrombin, the final protease in the coagulation cascade, is also FX by direct activation of FXI in the feedback mechanism.
It can contribute to Ia generation. Platelets, another important hemostatic component in whole blood, can be activated by thrombin and subsequently aid in FXIa formation. FXI-dependent amplification of thrombin production is due to activation of the thrombin-activated fibrinolytic inhibitor (TAFI).
Fibrin lysis (fibrin lysis) can be indirectly regulated. Therefore, FXI interacts with several components in the hemostatic system and plays an important role in blood coagulation and thrombosis (Gail).
ani & Rennes, supra; Emsley et al., Blood 2010, 115: 25
69-2577).

Coagulation factor XI (FXI) is a dimer composed of the same subunits of 80 kDa, with each subunit starting at the N-terminus consisting of four apple domains (A1, A2, A3 and A4) and a catalytic domain (A1, A2, A3 and A4). See FIG. 1B). FXI is a thymogen that circulates in a complex with high molecular weight kininogen (HK). HK binds to the A2 domain in FXI and is a physiological cofactor for FXIIa activation from FXI to FXIa. The remaining Apple domain in FXI also provides important physiological functions. For example, the FIX binding exosite is located at A3 and the FXIIa binding site is located at A4. F
Residues that are critical to XI dimerization are also located at A4 (Emsley et al., Supra).

While FXI has a relatively small contribution to hemostasis, it plays an important role in the pathological process of thrombus formation and has therefore been demonstrated by a wide variety of efforts in recent years to be a promising target for thrombosis. Has been done. Important data supporting this view are summarized in: (1) Ionis Pharmaceuticals I
nc. FXI Antisense Oligonucleotide (ASO) Phase II Clinical Trial (Bul)
Ler et al., N Engl J Med 2015, 372: 232-240)
FXI ASO showed a significant reduction in venous thromboembolism (VTE) with a lower bleeding tendency compared to enoxaparin in patients undergoing total knee arthroplasty. (2
) Human genetics and epidemiological studies (Duga et al., Semin Thromb Hemos)
t 2013; Chen et al., Drag Discov Today 2014; Key,
Hematology Am Soc Hematology Educ Program 2
014, 2014: 66-70), severe FXI deficiency (hemophilia C) results in a reduced risk of ischemic stroke and deep vein thrombosis, and conversely, elevated levels of FXI are VTE and ischemic. It has been shown to be associated with a higher risk of stroke. (3) A wide variety of preclinical studies have shown that FXI (a) inhibition or loss of function results in significant thrombosis prevention without interfering with hemostasis (Chen et al., Supra). Notably, the monoclonal antibodies 14E11 and 1A6 resulted in a significant reduction in thrombus in the hihi AV shunt thrombosis model (US Pat. No. 8,388,959; US Pat. No. 8,236,316; Tucker et al., Blood 2009, 113:
936-944; Cheng et al., Blood 2010, 116: 3981-3989)
.. In addition, 14E11 (which cross-reacts with mouse FXI) provided protection in an experimental model of acute ischemic stroke in mice (Lung et al., Transl Stroke).
Res 2012, 3: 381-389). Additional FXI-targeted mAbs have also been reported in preclinical models examining FXI as an antithrombotic target with minimal risk of bleeding (van Montfort et al., Thromb Haemost 2013, 110;
Takahashi et al., Thromb Res 2010, 125: 464-470; v
an Montfoort, Ph. et al. D. Thesis, University of A
msterdam, Amsterdam, Netherlands, 14 Novemb
er 2014). Therefore, inhibition of FXI is a promising strategy for novel antithrombotic therapies with improved benefit-risk profiles compared to current standard therapeutic anticoagulants.

Currently, there is a great unmet medical requirement for antithrombotic therapy in patients with severe renal disease or end-stage renal disease (ESRD). Approximately 650,000 patients in the United States have severe renal disease or ESRD, and these patients have an extremely high incidence of thrombotic and thromboembolic complications (MI, stroke / TIA, peripheral arterial disease (PAD)). ), Vascular access failure). ESRD patients are also more likely to exhibit bleeding events than the general population. Because no anticoagulant therapy of any kind is generally prescribed to ESRD patients (due to bleeding risk and lack of data on non-vitamin K antagonist oral anticoagulants (NOAC) in ESRD), acceptable benefits in these patients- Antithrombotic therapy with a risk profile is needed.

Brief Summary of the Invention The present invention is capable of selectively binding to the coagulation factor XI (anti-FXI antibody), preferably suppressing blood coagulation and related thrombosis without interfering with hemostasis. Provide a human antibody. The composition comprises an anticoagulant factor XI antibody capable of binding to certain epitopes of the apple 3 (A3) domain of coagulation factor XI. These antibodies are FXI
It exhibits neutralizing activity by inhibiting the conversion of the zymogen form FXI to its active coagulation factor FXIa by the action of Ia, and by inhibiting the FXIa-mediated activation of FIX. The antibody is useful for FXI inhibition, which can have clinically significant antithrombotic effects with a low risk of bleeding complications, and thus a more downstream coagulation factor,
It can result in an increased therapeutic index compared to, for example, FXa and thrombin. Therefore, these antibodies prevent thromboembolic complications, such as stroke prevention in atrial fibrillation (S).
Provides a therapeutic approach for PAF).

One of the untreated cohorts at risk of vascular thrombosis that could benefit from FXI inhibition is the severe and end-stage renal disease (ESRD) population, in which non-vitamin K antagonist oral anticoagulants due to bleeding concerns. Anticoagulants (NOACs) are typically not used, which leads to a lack of clinical trial experience. The antibodies in the present invention provide a novel anticoagulant therapy for the prevention of thrombotic complications in ESRD patients. The antibodies in the present invention may provide clinically significant antithrombotic effects with an acceptable risk of bleeding in ESRD patients.

In addition to ESRD and SPAF, FXI inhibition may also be indicated for additional patient populations at high risk of thrombosis. These include: 1) prevention of venous thromboembolism (VTE) and / or secondary prevention of VTE in orthopedic surgery; 2) reduction of major adverse limb events (MALE) in PAD and / or Reduction of revascularization; 3) Adjuvant therapy in ACS.

The present invention presents with six complementarity determining regions (CDRs) of anti-FXI antibodies of the αFXI-18623p family, αFXI-18611p family or αFXI-18611 family.
), Or one or more of the six CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof, αFXI-18623p family, αF.
Provided are antibodies or antigen-binding fragments of at least 6 complementarity determining regions (CDRs) of anti-FXI antibodies of the XI-18611p family or αFXI-18611 family, wherein the αFXI-18623 family of antibodies are sequenced. Number 28 or 29
An antibody of the αFXI-18611p family comprising a heavy chain (HC) variable region having the amino acid sequence shown in FIG. 30 and an LC variable region having the amino acid sequence shown in SEQ ID NO: 30 is designated as SEQ ID NO: 21 or 22. HC variable region having the amino acid sequence shown in
Includes a light chain (LC) variable region having the amino acid sequence set forth in SEQ ID NO: 25, αF
Antibodies of the XI-18611 family include an HC variable region having the amino acid sequence set forth in SEQ ID NO: 23 or 24 and an LC variable region having the amino acid sequence set forth in SEQ ID NO: 25. In a further embodiment, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the six CDRs are FXI-186.
CDR1, CDR2 and CDR3 of HC of anti-FXI antibodies of the 23p family, αFXI-18611p family or αFXI-18611 family, and FXI-1
8623p family, αFXI-18611p family or αFXI-18611
Containing or consisting of CDR1, CDR2 and CDR3 of the family LC
Here, the antibody of the αFXI-18623 family includes an HC variable region having the amino acid sequence shown in SEQ ID NO: 28 or 29 and an LC variable region having the amino acid sequence shown in SEQ ID NO: 30, and contains αFXI. The -18611p family of antibodies is SEQ ID NO: 2.
A heavy chain (HC) variable region having the amino acid sequence shown in 1 or 22 and SEQ ID NO: 2
Includes a light chain (LC) variable region having the amino acid sequence shown in 5 and αFXI-18.
Antibodies of the 611 family include an HC variable region having the amino acid sequence set forth in SEQ ID NO: 23 or 24 and an LC variable region having the amino acid sequence set forth in SEQ ID NO: 25. In a further embodiment, the antibody or antigen binding fragment is a coagulation factor XI.
It binds to the Apple 3 domain of (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the antibody or antigen binding fragment comprises an HC variable region having an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 21, 22, 23 and 24 and a sequence. L having the amino acid sequence shown in number 25
Includes a C variable region, where the HC variable region framework is 1, 2, 3, 4, 5, 6,
It is possible to include 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, and the LC variable region framework is 1, 2, 3, 4, 5, 6, 7, 8, It is possible to include 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

In a further aspect or embodiment of the invention, the antibody or antigen binding fragment comprises an HC variable region having an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 21, 22, 23 and 24 and a sequence. L having the amino acid sequence shown in number 25
Includes C variable region.

In a further aspect or embodiment of the invention, the antibody or antigen binding fragment is shown in SEQ ID NO: 30 with an HC variable region having an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 28 and 29. Containing an LC variable region having an amino acid sequence that has been identified, where the HC variable region framework is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, or deficiencies. It is possible to include loss or a combination thereof,
The LC variable region framework can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

In a further aspect or embodiment of the invention, the antibody or antigen binding fragment is shown in SEQ ID NO: 30 with an HC variable region having an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 28 and 29. Includes an LC variable region having the amino acid sequence.

In a further aspect or embodiment of the invention, the antibody is human IgG1, IgG2, I.
Contains heavy chain constant domains of the gG3 or IgG4 isotype. In a further aspect,
The constant domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. In certain embodiments, the constant domain is C.
It is possible to include terminal lysine or lack C-terminal lysine.

In a further aspect or embodiment of the invention, the antibody is human IgG1 or IgG4.
Contains isotyped heavy chain constant domains. In another embodiment, the heavy chain constant domain is of the IgG4 isotype and further contains a serine residue at position 228 (EU numbering) [which is serine at position 108 (position 108) of SEQ ID NO: 16 or 17. ) Corresponds to], including replacement with proline.

In a further aspect or embodiment of the invention, the antibody comprises an HC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16, 17, 18 or 19.

In a further aspect or embodiment of the invention, the antibody comprises a human kappa or lambda type light chain constant domain.

In a further aspect or embodiment of the invention, the antibody comprises an LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the antibody or antigen binding fragment is SEQ ID NO: 33, 35, 37, 39, 45, 47, 49, 51, 57, 59, 61, 6.
It comprises HC having an amino acid sequence selected from the group of amino acid sequences consisting of 3, 69, 71, 73 and 75 and LC having the amino acid sequence shown in SEQ ID NO: 26.

In a further aspect or embodiment of the invention, the antibody or antigen binding fragment is an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 41, 43, 53, 55, 65, 67, 77 and 79. Includes HC having the above and LC having the amino acid sequence set forth in SEQ ID NO: 31.

The present invention further comprises (a) a heavy chain (HC) having the amino acid sequence set forth in SEQ ID NO: 28.
The variable domain and the light chain (LC) variable domain having the amino acid sequence set forth in SEQ ID NO: 30, (b) the heavy chain (HC) variable domain having the amino acid sequence set forth in SEQ ID NO: 29 and SEQ ID NO: 30. The light chain (LC) variable domain having the indicated amino acid sequence, (b) the heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 21, and the amino acid sequence shown in SEQ ID NO: 25. Has a light chain (LC) variable domain, (
c) Heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 22 and light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25, (d) shown in SEQ ID NO: 23. A heavy chain (HC) variable domain having the amino acid sequence shown in the above and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25, or (e) the amino acid sequence shown in SEQ ID NO: 24. Provided is an antibody or antigen-binding fragment comprising a heavy chain (HC) variable domain having and a light chain (LC) variable domain having the amino acid sequence set forth in SEQ ID NO: 25.

In a further embodiment, the antibody or antigen binding fragment is coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation.

In certain embodiments, the HC and LC variable regions are 1, 2, 3, 4, 5, 6, 7,
It may include 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

In certain embodiments, the HC and LC constant domains are 1, 2, 3, 4, 5, 6,
It may include 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.
In certain embodiments, the constant domain can include a C-terminal lysine or can lack a C-terminal lysine.

In certain embodiments, the HC and LC variable regions are 1, 2, 3, 4, 5, 6, 7,
It can contain 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, and the HC and LC constant domains are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
It is possible to include individual amino acid substitutions, additions, deletions or combinations thereof. In certain embodiments, the constant domain can include a C-terminal lysine or can lack a C-terminal lysine.

In a further aspect or embodiment of the invention, the antibody further comprises SEQ ID NOs: 16, 17,
HC constant domain containing the amino acid sequence shown in 18 or 19, or 1, 2,
Includes variants thereof containing 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

In a further aspect or embodiment of the invention, the antibody further comprises an LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20, or 1, 2, 3, 4, 5, 6, 7, 8
, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

In another aspect or embodiment of the invention, the antibody or antigen binding fragment is shown in (a) a heavy chain (HC) variable domain having the amino acid sequence set forth in SEQ ID NO: 28 and SEQ ID NO: 30. It has a light chain (LC) variable domain having the amino acid sequence shown in the chain, (b) a heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 29, and an amino acid sequence shown in SEQ ID NO: 30. Light chain (LC) variable domain, (c
) Heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 21 and light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25, (d) shown in SEQ ID NO: 22. A heavy chain (HC) variable domain having the same amino acid sequence and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25, (e) SEQ ID NO: 2
A heavy chain (HC) variable domain having the amino acid sequence shown in 3 and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25, (f) the amino acid shown in SEQ ID NO: 24. A heavy chain (HC) variable domain having a sequence and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25, (g) HC variable region framework is 1, 2, 3, 4, 5, Variations of (a), (b), (c), (d), (e) or (f), including 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. Body, or (h) LC variable region framework 1, 2, 3, 4, 5, 6, 7, 8,
(A), (b) containing 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.
), (C), (d), (e), (f) or (g).

The present invention further describes (a) heavy chain complementarity determining regions (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 1, and HC-CDR having the amino acid sequence shown in SEQ ID NO: 2.
Heavy chain (HC) having variable domains and constant domains including HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 2 and (b) Heavy chain having the amino acid sequence shown in SEQ ID NO: 1. Complementarity determining regions (HC-CDR) 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and H having the amino acid sequence shown in SEQ ID NO: 4.
Heavy chain (HC) having a variable domain containing C-CDR3 and a constant domain, or (c)
Heavy chain complementarity determining regions (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 8.
, An antibody comprising a heavy chain (HC) having a variable domain including HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 9 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 10 and a constant domain. I will provide a. In a further embodiment, the antibody or antigen binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and FX.
Inhibits activation of I and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the antibody is human IgG1, IgG2, I.
Contains heavy chain constant domains of the gG3 or IgG4 isotype. In a further aspect,
The constant domain has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the amino acid sequence of the natural heavy chain constant domain of the human IgG1, IgG2, IgG3 or IgG4 isotype. , Additions, deletions or combinations thereof. In certain embodiments, the constant domain can include a C-terminal lysine or can lack a C-terminal lysine.

In a further aspect or embodiment of the invention, the antibody is human IgG1 or IgG4.
Contains isotyped heavy chain constant domains. In another embodiment, the heavy chain constant domain is of the IgG4 isotype and further contains a serine residue at position 228 (EU numbering) [which is serine at position 108 (position 108) of SEQ ID NO: 16 or 17. ) Corresponds to], including replacement with proline.

In a further aspect or embodiment of the invention, the antibody comprises an IgG4 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16 or 17. In another embodiment, the constant domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

In a further aspect or embodiment of the invention, the antibody comprises an IgG1 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 18 or 19. In a further embodiment, the constant domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

The present invention further
(A) Light chain complementarity determining regions (LC-) having the amino acid sequence shown in SEQ ID NO: 5.
CDR) 1, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 6 and a light chain (LC) having a variable domain and a constant domain containing LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 7. , Or (b) light chain complementarity determining regions (LC) having the amino acid sequence set forth in SEQ ID NO: 11.
-CDR) 1, a light chain (LC) having a variable domain and a constant domain containing LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 12 and LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 13. )
Provide an antibody or antigen-binding fragment containing. In a further embodiment, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the light chain (LC) is a human kappa light chain or a human lambda light chain, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. These include variants, including substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI), activating FXI and /. Alternatively, it inhibits factor XIa-mediated activation of factor IX. In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the antibody is an IgG4 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16 or 17, or 1, 2, 3, 4, ,.
It comprises a variant thereof comprising 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation. In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the antibody is an IgG1 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 18 or 19, or 1, 2, 3, 4, 5
, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof comprising a variant thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (FX).
I) binds to the Apple 3 domain of FXI activation and / or factor IX factor XI
a Inhibits mediated activation.

The present invention further
(A) Heavy chain complementarity determining regions (HC-) having the amino acid sequence shown in SEQ ID NO: 1.
CDR) 1, a heavy chain (HC) having a variable domain including HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 3 and a constant domain. , And (b) light chain complementarity determining regions having the amino acid sequence set forth in SEQ ID NO: 5 (LC-
CDR) 1, a light chain (LC) having a variable domain and a constant domain containing LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 6 and LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 7.
Provide an antibody or antigen-binding fragment containing. In a further embodiment, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the light chain is a human kappa light chain or a human lambda light chain, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.
These include variants, including additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and FXI.
And / or the factor XIa-mediated activation of factor IX. In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the antibody is IgG1, IgG2, Ig.
Heavy chain constant domain of G3 or IgG4 isotype, or native IgG1, IgG2
, 1, 2, 3, 4, 5, compared to the amino acid sequence of IgG3 or IgG4 isotype,
It comprises a variant thereof comprising 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (FXI).
) Binds to the Apple 3 domain and activates FXI and / or factor IX factor XIa
Inhibits mediated activation. In a further embodiment, the constant domain can include a C-terminal lysine or can lack a C-terminal lysine.

In a further aspect or embodiment of the invention, the antibody is human IgG1 or IgG.
Includes, 4 isotypes of heavy chain constant domains, or variants thereof, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. In, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX. In another embodiment, the heavy chain constant domain is of the IgG4 isotype and further contains a serine residue at position 228 (EU numbering) [which is SEQ ID NO: 16 or 17].
Corresponds to position 108 (serine at position 108)], including substitution with proline.

In a further aspect or embodiment of the invention, the antibody is an IgG4 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16 or 17, or 1, 2, 3, 4, ,.
It comprises a variant thereof comprising 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation.

In a further aspect or embodiment of the invention, the antibody is an IgG1 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 18 or 19, or 1, 2, 3, 4, ,.
It comprises a variant thereof comprising 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation.

The present invention further
(A) Heavy chain complementarity determining regions (HC-) having the amino acid sequence shown in SEQ ID NO: 1.
CDR) 1, a heavy chain (HC) having a variable domain including HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 4 and a constant domain. , And (b) light chain complementarity determining regions having the amino acid sequence set forth in SEQ ID NO: 5 (LC-
CDR) 1, a light chain (LC) having a variable domain and a constant domain containing LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 6 and LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 7.
Provide an antibody or antigen-binding fragment containing. In a further embodiment, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the light chain is a human kappa light chain or a human lambda light chain, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.
These include variants, including additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and FXI.
And / or the factor XIa-mediated activation of factor IX. In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the antibody is IgG1, IgG2, Ig.
Heavy chain constant domain of G3 or IgG4 isotype, or native IgG1, IgG2
, 1, 2, 3, 4, 5, compared to the amino acid sequence of IgG3 or IgG4 isotype,
It comprises a variant thereof comprising 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (FXI).
) Binds to the Apple 3 domain and activates FXI and / or factor IX factor XIa
Inhibits mediated activation. In a further embodiment, the constant domain can include a C-terminal lysine or can lack a C-terminal lysine.

In a further aspect or embodiment of the invention, the antibody is human IgG1 or IgG.
Includes, 4 isotypes of heavy chain constant domains, or variants thereof, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. In, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX. In another embodiment, the heavy chain constant domain is of the IgG4 isotype and further contains a serine residue at position 228 (EU numbering) [which is SEQ ID NO: 16 or 17].
Corresponds to position 108 (serine at position 108)], including substitution with proline.

In a further aspect or embodiment of the invention, the antibody is an IgG4 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16 or 17, or 1, 2, 3, 4, ,.
It comprises a variant thereof comprising 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation.

In a further aspect or embodiment of the invention, the antibody is an IgG1 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 18 or 19, or 1, 2, 3, 4, ,.
It comprises a variant thereof comprising 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation.

The present invention further
(A) Heavy chain complementarity determining regions (HC-) having the amino acid sequence shown in SEQ ID NO: 8.
CDR) 1, a heavy chain (HC) having a variable domain including HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 9 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 10 and a constant domain. , And (b) light chain complementarity determining regions (LC) having the amino acid sequence set forth in SEQ ID NO: 11.
-CDR) 1, a light chain (LC) having a variable domain and a constant domain containing LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 12 and LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 13. )
Provide an antibody or antigen-binding fragment containing. In a further embodiment, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the light chain is a human kappa light chain or a human lambda light chain, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.
These include variants, including additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and FXI.
And / or the factor XIa-mediated activation of factor IX. In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the antibody is IgG1, IgG2, Ig.
Heavy chain constant domain of G3 or IgG4 isotype, or native IgG1, IgG2
, 1, 2, 3, 4, 5, compared to the amino acid sequence of IgG3 or IgG4 isotype,
It comprises a variant thereof comprising 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (FXI).
) Binds to the Apple 3 domain and activates FXI and / or factor IX factor XIa
Inhibits mediated activation. In a further embodiment, the constant domain can include a C-terminal lysine or can lack a C-terminal lysine.

In a further aspect or embodiment of the invention, the antibody is human IgG1 or IgG.
Includes, 4 isotypes of heavy chain constant domains, or variants thereof, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. In, the antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX. In another embodiment, the heavy chain constant domain is of the IgG4 isotype and further contains a serine residue at position 228 (EU numbering) [which is SEQ ID NO: 16 or 17].
Corresponds to position 108 (serine at position 108)], including substitution with proline.

In a further aspect or embodiment of the invention, the antibody is an IgG4 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16 or 17, or 1, 2, 3, 4, ,.
It comprises a variant thereof comprising 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation.

In a further aspect or embodiment of the invention, the antibody is an IgG1 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 18 or 19, or 1, 2, 3, 4, ,.
It comprises a variant thereof comprising 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is a coagulation factor XI (F).
XI) binds to the Apple 3 domain, activates FXI and / or factor X of factor IX
Inhibits Ia-mediated activation.

In a further aspect or embodiment of the invention, the invention is a heavy chain complementarity determining region (HC) having (a) (i) the HC framework and the amino acid sequences set forth in SEQ ID NO: 8.
-CDR) 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 9 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 10; (ii) HC framework and SEQ ID NO: 1. Heavy chain complementarity determining regions (HC) having an amino acid sequence
-CDR) 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 3; (iii) shown in the HC framework and SEQ ID NO: 1. Heavy chain complementarity determining regions (HC) having an amino acid sequence
-CDR) 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 4; (iv) HC CDR1
, HC CDR2 or at least one of CDR3 has one, two or three amino acid substitutions,
Variants of (i), (ii) or (iii), including additions, deletions or combinations thereof;
Alternatively, (v) the HC framework comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, (i), (ii). , (Iii
) Or heavy chain (HC) having a variable domain containing a variant of (iv) and a constant domain;
(B) (i) Light chain complementarity determining regions (LC-CDR) 1 having the amino acid sequence shown in the LC framework and SEQ ID NO: 11, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 12. And LC-C having the amino acid sequence shown in SEQ ID NO: 13.
DR3; (ii) LC framework and light chain complementarity determining regions (LC-CDR) 1 having the amino acid sequence set forth in SEQ ID NO: 5, LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 6 and LC-CD having the amino acid sequence shown in SEQ ID NO: 7
R3; (iii) at least one of LC CDR1, LC CDR2 or LC-CDR3
One contains one, two or three amino acid substitutions, additions, deletions or combinations thereof, (i).
Or a variant of (ii); or (iv) LC framework 1, 2, 3, 4, 5,
Includes 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof,
A light chain (LC) having a variable domain containing a variant of (i), (ii) or (iii) and a constant domain; or an antibody comprising HC from (c) (a) and LC from (b). Where the antibody binds to the Apple 3 domain of coagulation factor XI (FXI) and FX
Inhibits activation of I and / or factor XIa-mediated activation of factor IX.

In a further aspect or embodiment of the invention, the HC constant domain is SEQ ID NO: 16,
The antibody according to claim 18, which comprises the amino acid sequence shown in 17, 18 or 19.

In a further aspect or embodiment of the invention, the antibody of claim 18 or 19, wherein the LC constant domain comprises the amino acid sequence set forth in SEQ ID NO: 20.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 33 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 35 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 45 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 47 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 49 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 51 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 59 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 61 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 63 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 69 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 33 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 71 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 73 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 75 and SEQ ID NO: 2.
An antibody comprising a light chain having the amino acid sequence shown in 6 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 39 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 41 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 43 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 53 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 55 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 57 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 65 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 67 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 69 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 77 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 79 and SEQ ID NO: 3.
An antibody comprising a light chain having the amino acid sequence shown in 1 is provided.

The present invention further relates to SEQ ID NOs: 33, 35, 37, 45, 47, 49, 51, 59, 61, 6
An antibody or SEQ ID NO: 39, 41, which comprises a heavy chain having the amino acid sequence set forth in 3, 69, 71, 73 or 75 and a light chain having the amino acid sequence set forth in SEQ ID NO: 26.
Cross-blocking an antibody containing a heavy chain having the amino acid sequence shown in 43, 53, 55, 57, 65, 67, 69, 77 or 79 and a light chain having the amino acid sequence shown in SEQ ID NO: 31. Provide an antibody or antigen-binding fragment that competes with the binding of the antibody.
Here, the antibody or antigen-binding fragment does not contain a mouse or rat amino acid sequence.

In another embodiment, the antibody or antigen binding fragment does not contain a non-human amino acid sequence.

In another embodiment, the antibody comprises (i) human IgG1 constant domain or variant or modified derivative thereof, or (ii) human IgG4 constant domain or variant or modified variant thereof.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG4 constant domain is at least at position 228 (E).
A variant containing a proline substitution of the serine residue at position 108 (U-numbered) or position 108 (as shown herein).

In another embodiment, the IgG1 or IgG4 constant domain is at least a mutant lacking a C-terminal lysine.

In another embodiment, the antibody or antigen binding fragment comprises a variable domain sequence comprising a framework characteristic of a human antibody.

The present invention further relates to SEQ ID NOs: 33, 35, 37, 45, 47, 49, 51, 59, 61, 6
An antibody or SEQ ID NO: 39, 41, which comprises a heavy chain having the amino acid sequence set forth in 3, 69, 71, 73 or 75 and a light chain having the amino acid sequence set forth in SEQ ID NO: 26.
Cross-blocking an antibody containing a heavy chain having the amino acid sequence shown in 43, 53, 55, 57, 65, 67, 69, 77 or 79 and a light chain having the amino acid sequence shown in SEQ ID NO: 31. Provided is a human antibody or antigen-binding fragment that competes with the binding of the antibody.

In another embodiment, the antibody or antigen binding fragment does not contain a non-human amino acid sequence.

In another embodiment, the antibody comprises (i) human IgG1 constant domain or variant or modified derivative thereof, or (ii) human IgG4 constant domain or variant or modified variant thereof.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG4 constant domain is at least at position 228 (E).
A variant containing a proline substitution of the serine residue at position 108 (U-numbered) or position 108 (as shown herein).

In another embodiment, the IgG1 or IgG4 constant domain is at least a mutant lacking a C-terminal lysine.

In another embodiment, the antibody or antigen binding fragment comprises a variable domain sequence comprising a framework characteristic of a human antibody.

The present invention further comprises a coagulation factor XI (FX) comprising the amino acid sequence YATRQFPSLEEHRNICL (SEQ ID NO: 82) and the amino acid sequence HTQTGTPTRITKL (SEQ ID NO: 83).
I) Provide an antibody or antigen-binding fragment that binds to an epitope on the above, where the antibody or antigen-binding fragment does not contain a mouse or rat amino acid sequence. In certain embodiments, binding to the epitope is determined by hydrogen-deuterium exchange mass spectrometry.

In another embodiment, the antibody or antigen binding fragment does not contain a non-human amino acid sequence.

In another embodiment, the antibody comprises (i) human IgG1 constant domain or variant or modified derivative thereof, or (ii) human IgG4 constant domain or variant or modified variant thereof.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, an IgG1 or IgG4 constant domain is a variant comprising at least 1, 2, 3 or 4 amino acid substitutions, additions, deletions or combinations thereof.

In another embodiment, the IgG4 constant domain is at least at position 228 (E).
A variant containing a proline substitution of the serine residue at position 108 (U-numbered) or position 108 (as shown herein).

In another embodiment, the IgG1 or IgG4 constant domain is at least a mutant lacking a C-terminal lysine.

In another embodiment, the antibody or antigen binding fragment comprises a variable domain sequence comprising a framework characteristic of a human antibody.

The present invention further comprises a coagulation factor XI (FX) comprising the amino acid sequence YATRQFPSLEEHRNICL (SEQ ID NO: 82) and the amino acid sequence HTQTGTPTRITKL (SEQ ID NO: 83).
I) provides a human antibody or antigen-binding fragment that binds to an epitope on the above, wherein the antibody is (i) a human IgG1 constant domain or a variant or modified derivative thereof, or (ii) a human IgG4 constant domain or Includes the variant or modified variant.
In certain embodiments, binding to the epitope is determined by hydrogen-deuterium exchange mass spectrometry.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG4 constant domain is at least at position 228 (E).
A variant containing a proline substitution of the serine residue at position 108 (U-numbered) or position 108 (as shown herein).

In another embodiment, the IgG1 or IgG4 constant domain is at least a mutant lacking a C-terminal lysine.

In another embodiment, the antibody or antigen binding fragment comprises a variable domain sequence comprising a framework characteristic of a human antibody.

The present invention further provides an isolated nucleic acid molecule encoding a light chain variable domain or a heavy chain variable domain of either the antibody or antigen binding fragment.

The present invention further comprises a coagulation factor XI (FX) comprising the amino acid sequence YATRQFPSLEEHRNICL (SEQ ID NO: 82) and the amino acid sequence HTQTGTPTRITKL (SEQ ID NO: 83).
I) provides a humanized antibody or antigen-binding fragment that binds to an epitope on the above, wherein the antibody is (i) a human IgG1 constant domain or a variant or modified derivative thereof.
Alternatively, (ii) includes a human IgG4 constant domain or a variant or modified variant thereof. In certain embodiments, binding to the epitope is determined by hydrogen-deuterium exchange mass spectrometry.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG1 or IgG4 constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. It is a mutant.

In another embodiment, the IgG4 constant domain is at least at position 228 (E).
A variant containing a proline substitution of the serine residue at position 108 (U-numbered) or position 108 (as shown herein).

In another embodiment, the IgG1 or IgG4 constant domain is at least a mutant lacking a C-terminal lysine.

In another embodiment, the antibody or antigen binding fragment comprises a variable domain sequence comprising a framework characteristic of a human antibody.

The present invention further provides an isolated nucleic acid molecule encoding a light chain variable domain or a heavy chain variable domain of either the antibody or antigen binding fragment.

The present invention further provides a composition comprising an antibody or antigen-binding fragment of any of the antibodies or antigen-binding fragments and a pharmaceutically acceptable carrier or diluent.

The invention further provides a method of treating a thromboembolic disorder or disease in a subject, comprising administering to the subject an effective amount of any antibody or antigen-binding fragment of either of the antibodies or antigen-binding fragments.

The present invention further comprises administering to a subject in need of treatment of a thromboembolic disorder or disease an effective amount of an antibody or antigen-binding fragment of any of the antibodies or antigen-binding fragments. Provide a method of treating a disorder or disease.

The present invention further provides the use of either the antibody or the antibody of the antigen-binding fragment for the manufacture of a medicament for treating a thromboembolic disorder or disease.

The present invention further provides antibodies for either the antibodies or antigen-binding fragments for the treatment of thromboembolic disorders or diseases.

The present invention further describes (i) heavy chain complementarity determining regions (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 1, and HC-CDR having the amino acid sequence shown in SEQ ID NO: 2.
A heavy chain having a variable domain and a constant domain constituting a heavy chain containing HC-CDR3 having the amino acid sequence shown in 2 and SEQ ID NO: 3 or 4, and (ii) the amino acid shown in SEQ ID NO: 5. Variables including light chain complementarity determining regions (LC-CDR) 1 with sequence, LC-CDR2 with amino acid sequence set forth in SEQ ID NO: 6 and LC-CDR3 having amino acid sequence set forth in SEQ ID NO: 7. A method for producing an antibody or antigen-binding fragment containing a light chain having a domain and a constant domain is provided, and the production method comprises a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain. It involves preparing the cells and culturing the host cells under conditions sufficient to produce the antibody or antigen-binding fragment and for a time sufficient to produce it.

In a further aspect or embodiment of the invention, the antibody is IgG1, IgG2, IgG.
Includes heavy chain constant domains of 3 or IgG4 isotypes.

In a further aspect or embodiment of the invention, the antibody comprises a heavy chain constant domain of IgG4 isotype.

In a further aspect or embodiment of the invention, the antibody is said to be SEQ ID NOs: 16, 17, 18.
Alternatively, it comprises a heavy chain constant domain comprising the amino acid sequence shown in 19.

In a further aspect or embodiment of the invention, the light chain comprises a human kappa light chain or a human lambda light chain.

In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the host cell is a Chinese hamster ovary cell or a human fetal kidney 293 cell.

In a further aspect or embodiment of the invention, the host cell is a yeast or filamentous fungal cell.

The present invention further describes (i) heavy chain complementarity determining regions (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 1, and HC-CDR having the amino acid sequence shown in SEQ ID NO: 2.
A heavy chain having a variable domain and a constant domain constituting a heavy chain containing HC-CDR3 having the amino acid sequence shown in 2 and SEQ ID NO: 3 or 4, and (ii) the amino acid shown in SEQ ID NO: 5. Variables including light chain complementarity determining regions (LC-CDR) 1 with sequence, LC-CDR2 with amino acid sequence set forth in SEQ ID NO: 6 and LC-CDR3 having amino acid sequence set forth in SEQ ID NO: 7. A method for producing an antibody or antigen-binding fragment containing a light chain having a domain and a constant domain is provided, and the production method comprises a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain. It involves preparing the cells and culturing the host cells under conditions sufficient to produce the antibody or antigen-binding fragment and for a time sufficient to produce it.

In a further aspect or embodiment of the invention, the antibody is IgG1, IgG2, IgG.
Includes heavy chain constant domains of 3 or IgG4 isotypes.

In a further aspect or embodiment of the invention, the antibody comprises a heavy chain constant domain of IgG4 isotype.

In a further aspect or embodiment of the invention, the antibody is said to be SEQ ID NOs: 16, 17, 18.
Alternatively, it comprises a heavy chain constant domain comprising the amino acid sequence shown in 19.

In a further aspect or embodiment of the invention, the light chain comprises a human kappa light chain or a human lambda light chain.

In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the host cell is a Chinese hamster ovary cell or a human fetal kidney 293 cell.

In a further aspect or embodiment of the invention, the host cell is a yeast or filamentous fungal cell.

(I) Heavy chain complementarity determining regions (HC-C) having the amino acid sequence shown in SEQ ID NO: 1.
DR) 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2, HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 3 or 4, or SEQ ID NO: 8
A heavy chain variable domain comprising HC-CDR1 having the amino acid sequence shown in, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 9 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 10. And (ii) the light chain complementarity determining regions (LC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 5, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 6, and SEQ ID NO: 7. L with the amino acid sequence shown
C-CDR3, or LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 11.
, LC-CDR2 and SEQ ID NO: 13 having the amino acid sequence set forth in SEQ ID NO: 12.
Provided is a method for producing an antibody or antigen-binding fragment containing a light chain variable domain containing LC-CDR3 having the amino acid sequence shown in the above, wherein the production method comprises a nucleic acid molecule encoding the heavy chain and the light chain. A host cell containing a strand-encoding nucleic acid molecule is prepared and the host cell is cultured under conditions sufficient to produce the antibody or antigen-binding fragment and for a time sufficient to produce it. Including.

In a further aspect or embodiment of the invention, the antibody is IgG1, IgG2, IgG.
Includes heavy chain constant domains of 3 or IgG4 isotypes.

In a further aspect or embodiment of the invention, the antibody comprises a heavy chain constant domain of IgG4 isotype.

In a further aspect or embodiment of the invention, the antibody is said to be SEQ ID NOs: 16, 17, 18.
Alternatively, it comprises a heavy chain constant domain comprising the amino acid sequence shown in 19.

In a further aspect or embodiment of the invention, the light chain comprises a human kappa light chain or a human lambda light chain.

In a further aspect or embodiment of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In a further aspect or embodiment of the invention, the host cell is a Chinese hamster ovary cell or a human fetal kidney 293 cell.

In a further aspect or embodiment of the invention, the host cell is a yeast or filamentous fungal cell.

The present invention further provides a composition comprising any of the above antibodies and a pharmaceutically acceptable carrier. In certain embodiments, the composition comprises an antibody comprising a heavy chain having a C-terminal lysine and a C.
Contains a mixture with an antibody containing a heavy chain lacking terminal lysine. In certain embodiments, the composition comprises an antibody disclosed herein in which the primary antibody form comprises a heavy chain having a C-terminal lysine. In certain embodiments, the composition comprises an antibody disclosed herein in which the primary antibody form comprises a heavy chain lacking a C-terminal lysine. In certain embodiments, the composition comprises an antibody disclosed herein, wherein approximately 100% of the antibody in the composition comprises a heavy chain lacking a C-terminal lysine.

Definitions As used herein, "antibody" means complete immunoglobulin, including recombinant production forms.
Includes any form of antibody that exhibits the desired biological activity. Therefore, it is used in the broadest sense, in particular monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, bipara. Includes, but is not limited to, topical (biparatopic) and chimeric antibodies. A "parent antibody" is an antibody obtained by exposure of the immune system to an antibody prior to modification of the antibody for its intended use (eg, humanization of the antibody for use as a human therapeutic antibody). is there.

"Antibody", in one embodiment, means a glycoprotein or an antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region ( _{abbreviated as VH} in the present specification) and a heavy chain constant region. In some naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is composed of three domains, namely CH1, CH2 and CH3. In some naturally occurring antibodies, each light chain is composed of a light chain variable region ( _{abbreviated herein as VL} ) and a light chain constant region. The light chain constant region is composed of one domain, namely CL. The V _H and _VL regions are the framework regions (F).
It can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called R). Each V _H and _VL has 3 CDRs and 4 FRs
They are located in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can result in binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q).

In general, the basic antibody structural unit comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain is approximately 10 that predominantly results in antigen recognition.
Includes variable regions of 0-110 or more amino acids. The carboxy-terminal portion of the heavy chain can define a constant region that primarily provides effector function. Typically, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are typically mu, delta,
Classified as gamma, alpha or epsilon, IgM, IgD, IgG, respectively
Determine antibody isotypes as IgA and IgE. In the light and heavy chains, the variable and constant regions are linked by the "J" region of about 12 or more amino acids, and the heavy chain also includes the "D" region of about 10 or more amino acids. Overall, Fundamen
tal Immunology Ch. 7 (Paul, W. ed., 2nd ed. Rave
n Press, N. et al. Y. (1989)).

The heavy chain of the antibody can contain or not contain terminal lysine (K), or terminal glycine and lysine (GK). Thus, a particular embodiment of an antibody in the invention comprising a heavy chain constant region amino acid sequence shown herein that lacks a terminal lysine but terminates with a glycine residue comprises an embodiment in which no terminal glycine residue is also present. Further included. This is because terminal lysine, and sometimes glycine and lysine, are cleaved together during antibody expression.

As used herein, an "antigen-binding fragment" is a fragment of an antibody, i.e.
An antibody fragment, which possesses the ability to specifically bind to an antigen bound by a full-length antibody,
For example, it means a fragment having one or more CDR regions. Examples of antibody-binding fragments include Fab, Fab', F (ab') ₂ and Fv fragments; diabodies;
Includes, but is not limited to, single chain antibody molecules such as sc-Fv; as well as multispecific antibodies and Nanobodies formed from antibody fragments.

As used herein, "Fab fragment" is one made up of the variable region and C _{H 1} of the light chain and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

As used herein, "Fab 'fragment" one light chain, and a V _H domain and C _H 1 one heavy chain portions or fragments containing ₁ domain and also,, C H ₁ and C _H 2 It contains interdomain regions, so that interchain disulfide bonds can be formed between the two heavy chains of the _two Fab'fragments to form two F (ab') molecules.

As used herein, "F (ab') ₂ fragment" refers to two light chains, as well as _CH 1.
And C _{H 2} contains two heavy chains containing moieties and V _H domains of the constant region between the domains, so that interchain disulfide bond is formed between those two heavy chains. Thus, an F (ab') ₂ fragment is composed of two Fab'fragments linked by a disulfide bond between their two heavy chains. The "F (ab') ₂ fragment" can be the product of pepsin cleavage of the antibody.

As used herein, the "Fv region" includes variable regions from both heavy and light chains, but lacks constant regions.

These and other potential constructs are described in Chan & Carter (2010) Nat. R
ev. Immunol. It is described at 10:301. These antibody fragments
Obtained using conventional techniques known to those of skill in the art, the fragments are screened for usefulness as in the case of intact antibodies. Antigen-binding moieties can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulin.

"Fc" region as used herein, contains two heavy chain fragments comprising a C _{H 1} and C _{H 2} domains of an antibody. Two heavy chain fragments thereof linked by a disulfide bond 2 or more, and hydrophobic interactions C _{H 3} domain.

As used herein, "diabody" means a small antibody fragment with two antigen binding sites, which fragment is a light chain variable domain ( _VL ) within the same polypeptide chain.
Includes heavy chain variable domains ( _VH ) ( _VH - _VL or _VL - _{VH) linked to.} By using a linker that is too short to allow pairing between two domains on the same strand, those domains are forced to pair with complementary domains on another strand, and the two domains. Generates an antigen binding site. The diabodies are, for example, EP 404,097; WO 93/1.
1161; and Hollinger et al. (1993) Proc. Natl. Acad S
ci. USA 90: 6444-6448, which is described in more detail. A review of engineered antibody variants generally includes Holliger and Holliger (2005).
) Nat. Biotechnol. 23: 1126-1136.

As used herein, a "bispecific antibody" is an artificial hybrid antibody that has two different heavy / light chain pairs and thus two different binding sites. For example, bispecific antibodies are antibodies αFXI-13654p, αFXI-13716p or αFXI-13716.
First antibody or variant embodiment comprising at least 6 CDRs of (where one or more of the above 6 CDRs has 1, 2 or 3 amino acid substitutions, additions, deletions or combinations thereof. ), A first heavy chain / light chain pair containing one light chain, and one heavy chain and one light chain of a second antibody having specificity for an antigen of interest other than FXI. Can be included with a second heavy chain / light chain pair. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab'fragments. For example, Songsivilai et al. (1)
990) Clin. Exp. Immunol. 79: 315-321; Kostelny
Et al. (1992) J. et al. Immunol. See 148, 1547-1553. In addition, the bispecific antibody is "diabody" (Holliger et al. (1993) PNAS USA
90: 6444-6448) or "Janusin" (Traune)
cker et al. (1991) EMBO J. et al. 10: 3655-3459 and Traunec
ker et al. (1992) Int. J. Cancer Super. It can be formed as 7: 51-52).

The "isolated" antibodies or antigen-binding fragments thereof used herein are at least partially free of the cells from which they were produced or other biological molecules from the cell culture. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other substances such as cell residues and growth media. The isolated antibody or antigen-binding fragment can also be at least partially free of expression system components such as biological molecules from the host cell or in its growth medium. In general, the term "isolated" refers to the complete absence of such biological molecules, or the absence of water, buffers or salts.
Alternatively, it does not refer to an ingredient of a pharmaceutical preparation containing the antibody or fragment.

As used herein, "monoclonal antibody" means a substantially homogeneous population of antibodies, i.e., except for possible spontaneous mutations in which the antibody molecules that make up the population may be present in small amounts. It is the same in the amino acid sequence. In contrast, conventional (polyclonal) antibody preparations typically include a number of different antibodies with different amino acid sequences in variable domains that are often specific for different epitopes. The modifier "monoclonal" indicates the property of the antibody to be obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibody used in accordance with the present invention is Kohler et al. (1975) Nature 2.
It can be produced by the hybridoma method first described by 56,495, or by the recombinant DNA method (see, eg, US Pat. No. 4,816,567). Further, "monoclonal antibody" is, for example, Clackson et al. (1991).
) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Mol.
It can be isolated from the phage antibody library using the techniques described in Biol 222: 581-597. Presta (2005) J. et al. Allergy Clin. I
mmunol. See also 116: 731.

The "chimeric antibody" used herein is an antibody having a variable domain from the first antibody and a constant domain from the second antibody, wherein (i) the first antibody and the second antibody are different species. From (US Pat. No. 4,816,567 and Morrison et al., (19).
84) Proc. Natl. Acad. Sci. USA 81: 6851-6855),
Or (ii) the first and second antibodies are from different isotypes (eg, variable domain from IgG1 antibody and constant domain from IgG4 antibody, eg α
FXI-13465p-IgG4 (S228P)). In one embodiment, the variable domain is obtained from a human antibody (“parent antibody”) and the constant domain sequence is a non-human antibody (eg, for example).
Obtained from mice, rats, dogs, monkeys, gorillas, horses). In another embodiment, the variable domain is obtained from a non-human antibody (“parent antibody”) (eg, mouse, rat, dog, monkey, gorilla, horse) and the constant domain sequence is obtained from a human antibody. In another embodiment, the variable domain is obtained from a human IgG1 antibody (“parent antibody”) and the constant domain sequence is obtained from a human IgG4 antibody.

As used herein, "humanized antibody" means the form of an antibody that contains sequences from both human and non-human (eg, mouse or rat) antibodies. In general, a humanized antibody comprises at least one, typically two, all of the variable domains, where the hypervariable loop corresponds to that of a non-human immunoglobulin and all or all of the framework (FR) regions. Virtually all are of the human immunoglobulin sequence. The humanized antibody may optionally contain at least a portion of the human immunoglobulin constant region (Fc).

As used herein, "fully human antibody" is a human immunoglobulin amino acid sequence or a variant thereof sequence (recombinant to obtain a fully human antibody having a modified function or potency as compared to an antibody lacking a mutation. It means an antibody containing the mutation introduced therein.
Fully human antibodies do not contain non-human immunoglobulin amino acid sequences, eg, constant and variable domains (including CDRs) include human sequences other than those resulting from the mutations described above. Full human antibodies are fully human antibody libraries in which diversity is generated in the library in silico (eg, US Pat. No. 8,877,688 or 8,691,730).
Can include the amino acid sequence of an antibody or immunoglobulin obtained from (see No.).
Fully human antibodies include such antibodies produced in non-human organisms, for example, mouse carbohydrates when fully human antibodies are produced in mice, in mouse cells, or in hybridomas derived from mouse cells. May contain chains. Similarly, "mouse or murine antibody" means an antibody that contains only mouse or murine immunoglobulin sequences. Alternatively, fully human antibodies may contain rat carbohydrate chains when produced in rats, in rat cells, or in hybridomas derived from rat cells. Similarly, "rat antibody" means an antibody that contains only rat immunoglobulin sequences.

As used herein, the "non-human amino acid sequence" for an antibody or immunoglobulin is referred to as.
It means an amino acid sequence characteristic of the amino acid sequence of a non-human mammal. The term is an antibody or antibody obtained from a fully human antibody library in which incilico produces diversity in the library (see, eg, US Pat. No. 8,877,688 or 8,691,730). Does not contain immunoglobulin amino acid sequences.

As used herein, "effector function" means biological activity resulting from the Fc region of an antibody, which varies by antibody isotype. Examples of antibody effector function include Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (CDC).
ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors); as well as B cell activation.

The variable region of each light / heavy chain pair forms an antibody binding site. Therefore, in general, intact antibodies have two binding sites. Except for bifunctional or bispecific antibodies, their two binding sites are generally the same.

Typically, both heavy and light chain variable domains have three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). Including. CDRs are usually aligned by framework regions to allow binding to specific epitopes. Generally, from the N-terminus to the C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acid attribution to each domain is generally defined by Sequences of Prot.
eins of Immunological Interest, Kabat et al .; Na
torsional Institutes of Health, Bethesda, Md.
; ⁵ th ed. NIH Publ. No. 91-3242 (1991); Kabat
(1978) Adv. Prot. Chem. 32: 1-75; Kabat et al. (1977)
J. Biol. Chem. 252: 6609-6616; Chothia et al. (1987)
J Mol. Biol. 196: 901-917 or Chothia et al. (1989) N
It is based on the definition of ature 342: 878-883.

As used herein, "hypervariable region" means an amino acid residue of an antibody that results in antigen binding. Hypervariable regions are "complementarity determining regions" or "CDRs" (ie, CDRL1, CDRL2 and CDRL3 within the light chain variable domain and CDRH within the heavy chain variable domain.
1. Contains amino acid residues from CDRH2 and CDRH3). Kabat et al. (1991)
) Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, Na
torsional Institutes of Health, Bethesda, Md.
See (the sequences define the CDR regions of the antibody). Also, Chothia and Lesk (1987) J. Mol. Mol. Biol. See also 196: 901-917 (structure defines the CDR regions of the antibody).

As used herein, the "framework" or "FR" residue means a variable domain residue other than the hypervariable region residue defined herein as a CDR residue.

As used herein, "conservatively modified variants" or "conservative substitutions" refer to amino acids having similar properties (eg, charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and stiffness). It means that it is replaced with another amino acid having (such as), where the change can often be made without altering the biological activity of the protein. In general, one of ordinary skill in the art recognizes that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (eg, Watson et al. (1987) Molecular Biol).
ogy of the Gene, The Benjamin / Cummings Pu
b. Co. , P. 224 (see 4th Ed.)). Also, structurally or functionally similar amino acid substitutions are unlikely to impair biological activity. Typical conservative substitutions are shown in the table below.

As used herein, the term "epitope" or "antigen determinant" means a site on an antigen (eg, FXI) to which an immunoglobulin or antibody specifically binds. Epitopes within protein antigens can be formed from both continuous (adjacent) amino acids (usually linear epitopes) or discontinuous amino acids juxtaposed by tertiary folding of proteins (usually conformational epitopes). Epitopes formed from continuous amino acids are typically, but not always, retained upon exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically in a denaturing solvent. It disappears when processed. Epitopes are typically at least 3 in a unique spatial conformation.
Contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. A method for determining which epitope a given antibody binds to (ie,
Epitope mapping is well known in the art and includes, for example, immunoblotting and immunoprecipitation assays, in which overlapping or continuous peptides (eg, from FXI) are given antibodies (eg, from FXI). For example, it is tested for reactivity with anti-FXI antibody). Methods for determining the spatial conformation of epitopes include techniques in the art and techniques described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance and HDX-MS ( For example, Epitope Mapping P
rotocols in Methods in Molecular Biology
, Vol. 66, G. E. See Morris, (1996)).

The term "epitope mapping" refers to a method of identifying molecular determinants on an antigen involved in antibody-antigen recognition.

The term "binding to the same epitope" for two or more antibodies means that the antibody binds to the same segment of amino acid residue as determined by a given method.
Techniques for determining whether an antibody binds to an "identical epitope on FXI" with an antibody described herein will provide, for example, an epitope mapping method, eg, the atomic resolution of an epitope. Antigen: X-ray analysis of the crystals of the antibody complex and hydrogen / deuterium exchange mass analysis (HDX-MS) are included. There are other methods of monitoring antibody binding to antigen fragments (eg, proteolytic fragments) and antibody binding to mutants of the antigen, in which case loss of binding due to modification of amino acid residues in the antigen sequence. Is often regarded as an indicator of epitope components (eg, alanin scanning mutagenesis-Cunningha).
m & Wells (1985) Science 244: 1081). Computer-based combinatorial methods for epitope mapping can also be used. These methods are based on the ability of the antibody of interest to affinity-separate a particular short peptide from the combinatorial phage display peptide library.

An antibody that "competes with another antibody for binding to a target such as FXI" means an antibody that (partially or completely) inhibits the binding of said other antibody to that target. Whether the two antibodies compete with each other for binding to the target, i.e. whether one antibody inhibits the binding of the other antibody to the target and the degree of inhibition is determined using known competing experiments. sell. In certain embodiments, the antibody is at least 10%, 20%, 30%, 40%, 50.
%, 60%, 70%, 80%, 90% or 100% compete with and inhibit the binding of another antibody to the target. The level of inhibition or competition is which antibody is a "blocking antibody" (ie,
It can depend on whether it is the cold antibody) that is first incubated with the target. Competitive assays include, for example, Ed Harlow and David Lane,
Cold Spring Harb Protocol; 2006; doi: 10.1101
/ Pdb. prot4277 or "Using Antibodies" (Ed Ha)
low and David Lane, Cold Spring Harbor Lab
orientation Press, Cold Spring Harbor, N. et al. Y. ， US
It can be done as described in Chapter 11 of A 1999). Competitive antibodies are identical, overlapping or adjacent epitopes (eg, those demonstrated by steric hindrance).
Combine with.

Other competitive binding assays include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al., Methods in Enzymology 9:24).
2 (1983)); solid phase direct biotin-avidin EIA (Kirklan)
d et al., J. Mol. Immunol. 137: 3614 (1986)); solid phase direct labeling assay, solid phase direct labeling sandwich assay (Hallow and Lane, An).
tibodies: A Laboratory Manual, Cold Spring
See Harbor Press (1988)); Solid phase direct labeling with 1-125 labeling RIA (Morel et al., Mol. Immunol. 25 (1): 7 (1988).
); Solid phase direct biotin-avidin EIA (Chung et al., Virol)
Ogy 176: 546 (1990); and direct labeling RIA (Moldenhaue)
r et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, "specifically binds" to an antigen such as FXI means that the antibody or other ligand is totally or partially associated with FXI preferentially and is another molecule. In particular, it means that it does not associate with molecules found in human blood or serum. Antibodies ^typically with high affinity, represented by the 10 -7 ^{to 10 -11} M or less the dissociation constant _{(K D),} specifically binds to its cognate (cognate) antigen. Any _{K D} of greater than about ^{10 -6} M are also considered to indicate nonspecific binding. As used herein, "specifically binds" to an antigen antibody, ^{10 -7} M or less for _{K D,} ^{10 -8} M or less or of 5 × ^{10 -9} M or less in certain embodiments, or 10 with high affinity to mean having a ^-8 M to -11 ^M or less of K _D, binds to the antigen and substantially the same antigen, is a high affinity for an irrelevant antigen refers to an antibody that does not bind. The kinetics of coupling can be measured by the surface plasmon resonance described in Example 1 herein.

An antigen can be said to be "substantially identical" to a given antigen if it exhibits a high degree of amino acid sequence identity to the given antigen, for example, it is given. At least 80%, at least 90%, at least 95% of the amino acid sequence of the antigen
, At least 97% or at least 99% or more amino acid sequence identity. For example, antibodies that specifically bind to human FXI are also non-human primate species (
For example, it may cross-react with FXI from cynomolgus monkeys) but not with FXI from other species or antigens other than FXI.

As used herein, the "isolated nucleic acid molecule" is not accompanied by all or part of the polynucleotide if the isolated polynucleotide is found in nature, or it is not naturally linked. Means genomic, mRNA, cDNA or synthetically derived DNA or RNA or any combination thereof linked to a polynucleotide. For the purposes of the present disclosure, it should be understood that a "nucleic acid molecule" containing a particular nucleotide sequence does not contain an intact chromosome. An isolated nucleic acid molecule that "contains" the specified nucleic acid sequence is, in addition to the specified sequence, up to 10 or even 20 or more of other proteins or parts or fragments thereof. It can contain a coding sequence, or it can contain a functionally linked regulatory sequence that controls the expression of the coding region of the listed nucleic acid sequence, and / or it contains a vector sequence. It is possible.

As used herein, "therapeutic" or "therapeutic" refers to a therapeutic agent, such as a composition containing either an antibody or an antigen-binding fragment of the invention, wherein the therapeutic agent has therapeutic or prophylactic activity. Means to be administered internally or externally to a subject or patient who has or is suspected of having one or more of the symptoms of the indicated disease. Typically, the therapeutic agent is one or more disease symptoms in a treatment subject or population by inducing regression of such symptoms or suppressing the progression of such symptoms to any clinically measurable degree. It is administered in an amount effective to alleviate the disease. The amount of therapeutic agent effective in alleviating any particular disease symptom can vary depending on factors such as the patient's condition, age and weight, and the ability of the drug to elicit the desired response in the subject. Whether the disease symptoms are alleviated can be assessed by any clinical scale typically used by physicians or other skilled health care providers to assess the severity or progression of the symptoms. The term also delays the onset of disability-related symptoms and /
Or include reducing the severity of symptoms of such disorders. The term further includes amelioration of existing uncontrolled or undesired symptoms, prevention of additional symptoms, and amelioration or prevention of the underlying cause of such symptoms. Therefore, the term has provided beneficial results to human or animal subjects with a disorder, disease or condition, or to a human or animal subject who may develop such a disorder, disease or condition. Is shown.

As used herein, "treatment" means therapeutic treatment and diagnostic application when applied to a human or veterinary subject. "Treatment" includes contacting a human or animal subject with an antibody or antigen-binding fragment of the invention, where it applies to a human or veterinary subject.

As used herein, "therapeutically effective amount" means the amount of a particular substance sufficient to achieve the desired effect in the subject being treated. For example, this may be the amount required to suppress coagulation for at least 192 to 288 hours as determined by the aPTT assay, or the amount required to suppress FXI activation. When administered to a subject, a dose that achieves a target tissue concentration that has been shown to achieve the desired in vitro effect is commonly used.

As used herein, "thrombosis" refers to an intravascular clot ("thrombosis" that impedes the flow of blood through the circulatory system.
It means the formation or presence of (also called a thrombus). Thrombosis is usually caused by abnormalities in blood composition, quality of blood vessel walls and / or properties of blood flow. Clot formation is often
It is caused by damage to the walls of blood vessels (such as due to trauma or infection) and slowing or stagnation of blood flow beyond the point of damage. In some cases, abnormal coagulation causes thrombosis.

As used herein, "without interfering with hemostasis" means that little or no detectable bleeding is observed in a subject or patient after administration of the antibody or antibody fragment disclosed herein to the subject or patient. Means no. When targeting factor XI, inhibiting the conversion of factor XI to factor XIa or the activation of factor IX by factor XIa suppresses coagulation and related thrombosis without bleeding. In contrast, inhibiting the conversion or activity of factor XI suppresses coagulation, but also induces bleeding or increases the risk of bleeding.

Detailed Description of the Invention The present invention provides an anticoagulant factor XI antibody that binds to the Apple 3 domain of coagulation factor XI (FXI). These anti-FXI antibodies and antigen-binding fragments are inhibitors of FXI activation by factor XIIa and are useful in suppressing blood coagulation and related thrombosis (ie, antithrombotic indications) without interfering with hemostasis. .. For example, the anti-FXI antibody is used in the treatment and prevention of venous thromboembolism (VTE), stroke prevention in atrial fibrillation (SPAF),
Or certain medical device related thromboembolic disorders (eg, stents, intravascular stent grafts,
Catheter (heart or vein), continuous flow ventricular assist device (CF-LVADS), hemodialysis,
It can be used for the treatment and prevention of cardiopulmonary bypass and extracorporeal oxygenation (ECMO), ventricular assist device (VADS)). Therefore, the anti-FXI antibodies disclosed herein are useful in providing therapy for treating thromboembolic disorders or disorders in patients or subjects in need of such therapy.

FXI is a homodimeric serine protease having an essential component of the domain structure and the endogenous pathway of the coagulation cascade shown in FIG. 1B. FXI thymogen is factor XIIa
Can be cleaved into its activated form, FXIa. FXIa then activates factor IX, ultimately inducing thrombin production and clot formation. The anti-FXI antibodies disclosed herein inhibit the conversion of FXI to FXIa (see Figure 1A).

Anti-FXI antibody molecules were obtained from the fully human synthetic IgG1 / kappa library presented on the surface of the engineered yeast strain. Human and NHP plasma kallikrein (a protein that exhibits 56% amino acid identity to FXI) or other human coagulation cascade proteins (proteins that can bind to human FXI and non-human primate (NHP) FXI with sub-nanomolar affinity, but FII // IIa, FVII / VIIa, FIX / IXa, FX / Xa and FXI
FX the library to identify antibodies that do not bind to I / XIIa)
Screened with I or FXIa. Two antibodies with these properties, ie
αFXI-18611p and αFXI-18623p were identified. These antibodies
It is a fully human antibody containing a human kappa (κ) light chain and a human IgG1 (γ1) isotype heavy chain. The antibody binds to an epitope of FXI thymogen comprising SEQ ID NOs: 82 and 83 located within the Apple 3 domain of FXI. In addition, these antibodies bind to FXIa with the same affinity as FXI thymogen.

Antibodies of the αFXI-18611p family have heavy chain (HC) complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
Includes 1, 2 and 3 and light chain (LC) CDRs 1, 2 and 3 having the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively. αFXI-18611
The p-family is a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 21 or 22.
) Includes an antibody comprising a variable domain and a light chain (LC) variable domain comprising the amino acid sequence of SEQ ID NO: 25.

Antibodies of the αFXI-18611 family have heavy chain (HC) complementarity determining regions (CDRs) 1 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
, 2 and 3 and light chain (LC) CDRs 1, 2 and 3 having the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The αFXI-18611 family comprises antibodies comprising a heavy chain (HC) variable domain comprising the amino acid sequence set forth in SEQ ID NO: 23 or 24 and a light chain (LC) variable domain comprising the amino acid sequence of SEQ ID NO: 25. ..

The αFXI-18623p family of antibodies are associated with HC CDR1, 2 and 3 having the amino acid sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively. Includes LC CDRs 1, 2 and 3 with the amino acid sequences shown. The αFXI-13716p family comprises antibodies comprising a heavy chain (HC) variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28 or 29 and a light chain (LC) variable domain comprising the amino acid sequence of SEQ ID NO: 30. .. Antibodies in this family were obtained from a different germline than the previous family.

The present invention further comprises an anti-FXI antibody containing at least 6 CDRs of an anti-FXI antibody of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family, or one or more of the above six CDRs being one or two. Alternatively, there are embodiments thereof having three amino acid substitutions, additions, deletions or combinations thereof, and methods of using the antibody for treating antithrombotic indications such as SPAF.

In certain embodiments, the anti-FXI antibody is the αFXI-18611p family, αFX.
H of anti-FXI antibody of I-18611 family or αFXI-18623p family
C variable domain or the HC variable domain is 1, 2, 3, 4, 5, 6, 7, 8, 9
Or at least a variant thereof containing 10 amino acid substitutions, additions, deletions or combinations thereof.

In certain embodiments, the anti-FXI antibody is the αFXI-18611p family, αFX.
L of anti-FXI antibody of I-18611 family or αFXI-18623p family
C variable domain or LC variable domain is 1, 2, 3, 4, 5, 6, 7, 8, 9
Or at least a variant thereof containing 10 amino acid substitutions, additions, deletions or combinations thereof.

In certain embodiments, the anti-FXI antibody is the αFXI-18611p family, αFX.
H of anti-FXI antibody of I-18611 family or αFXI-18623p family
C variable domain or the HC variable domain is 1, 2, 3, 4, 5, 6, 7, 8, 9
Or its variants, including 10 amino acid substitutions, additions, deletions or combinations thereof, and α
FXI-18611p family, αFXI-18611 family or αFXI-1
LC variable domain of 8623p family of anti-FXI antibodies, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. Includes at least its variants, including.

In certain embodiments, the antibodies in the invention are anti-FX of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
At least the embodiment of the I antibody, wherein one or more of the six CDRs, or one or more of the above six CDRs, has one, two or three amino acid substitutions, additions, deletions or combinations thereof.
In addition, it includes heavy chains (HC) that are of the human IgG1, IgG2, IgG3 or IgG4 isotypes, and light chains (LC) that can be of the kappa or lambda type. In other embodiments, the antibody is the αFXI-18611p family, αFXI-1861.
6 CDRs of 1 family or αFXI-18623p family of anti-FXI antibodies
Alternatively, one or more of the above six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof, and further comprises IgM, IgD.
, IgA or IgE class. In certain embodiments, human IgG
1, IgG2, IgG3 or IgG4 isotypes are 1, 2, 3, 4, 5, 6, 7, 8
, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

In certain embodiments, the antibody is αFXI-18611p family, αFXI.
Of the anti-FXI antibodies of the -18611 family or the αFXI-18623p family, 6
It is possible that the CDRs, or one or more of the six CDRs described above, include at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof. It is possible to include HC constant domains of the IgG4 isotype. The IgG4 framework results in antibodies that have little or no effector function. In another aspect of the invention, the antibody is an anti-F of the αFXI-18611p family, the αFXI-18611 family or the αFXI-18623p family.
It is possible to include at least six CDRs of the XI antibody, or at least one or more of the above six CDRs having one, two or three amino acid substitutions, additions, deletions or combinations thereof. Further, it is possible to include an HC constant domain of the IgG4 isotype fused to the HC variable domain of the IgG1 isotype. In another aspect of the invention, the antibody is the αFXI-18611p family, αFX.
H of anti-FXI antibody of I-18611 family or αFXI-18623p family
C variable domain and LC variable domain, or the HC and LC variable domain
Independently, it is possible to include 1, 2, 3, 4, 5, 6, 7, 8, 9 or its variants containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, and further. , HC constant domains of the IgG4 isotype can be included. In another aspect of the invention, the antibody is an HC variable domain and LC of an anti-FXI antibody of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family, or the HC and LC independently. 1, 2, 3, 4, 5, 6, 7, 8
, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof can be included and further can include HC constant domains of the IgG4 isotype.

The antibodies of the invention are further monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), biparatopics (bi).
Paratopic; includes fully human and chimeric antibodies, but
It is not limited to these.

In general, the heavy chain amino acid sequence of an antibody such as IgG1 or IgG4 has a lysine at the C-terminus of the heavy chain constant domain. In some cases, to improve the homogeneity of the antibody product
It is possible to produce antibodies lacking the C-terminal lysine. The anti-FXI antibody of the present invention is C.
It includes embodiments in which a terminal lysine is present and embodiments in which a C-terminal lysine is absent. For example, the IgG1 HC constant domain can have the amino acid sequence set forth in SEQ ID NO: 18 or 19, and the IgG4 HC constant domain can have SEQ ID NO: 16 or 17.
It is possible to have the amino acid sequence shown in.

In certain embodiments, the N-terminal amino acid of HC can be a glutamine residue. In certain embodiments, the N-terminal amino acid of HC can be a glutamic acid residue. In certain embodiments, the N-terminal amino acid is modified to be a glutamate residue.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided is an anti-FXI antigen binding fragment comprising at least one of the above six CDRs comprising one or two or three amino acid substitutions, additions, deletions or combinations thereof.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided is an anti-FXI Fab fragment comprising at least one of the above six CDRs comprising one or two or three amino acid substitutions, additions, deletions or combinations thereof.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
An anti-FXI antibody, wherein one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof, and an antigen binding fragment thereof comprising an Fc region. , As well as how to use them.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided is an anti-FXI Fab'fragment in which one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided is _{anti-FXI F (ab') 2} , wherein one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
An anti-FXI Fv fragment comprising at least one of the above six CDRs comprising one or two or three amino acid substitutions, additions, deletions or combinations thereof is provided.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided is an anti-FXI scFv fragment comprising at least one of the above six CDRs comprising one or two or three amino acid substitutions, additions, deletions or combinations thereof.

The present invention further comprises three HC CDRs or three LC CDRs of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family, or one or more of the HC or LC CDRs. Provided is an anti-FXI domain antibody comprising at least its embodiments having two or three amino acid substitutions, additions, deletions or combinations thereof. In one embodiment of the invention, the domain antibody is a single domain antibody or Nanobody. In one embodiment of the invention, the domain antibody is a CDR of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family, or one or more of the CDRs 1, 2
Or a Nanobody comprising at least its embodiment having three amino acid substitutions, additions, deletions or combinations thereof.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided is an anti-FXI divalent antibody comprising at least one of the above six CDRs comprising one or two or three amino acid substitutions, additions, deletions or combinations thereof.

The present invention further provides bispecific antibodies and antigen-binding fragments having binding specificity for FXI and other antigens of interest, and methods of their use.

Biparatopic antibodies are antibodies that have binding specificity for different epitopes on the same antigen. The present invention further relates to αFXI-1.
8611p family, αFXI-18611 family or αFXI-18623p
A first antibody of a family of anti-FXI antibodies comprising at least 6 CDRs, or an embodiment thereof in which one or more of the CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof. A biparatopic antibody having a first heavy / light chain pair and a second heavy / light chain pair of second antibodies having specificity for an FXI epitope different from the epitope recognized by the first heavy / light chain pair. provide.

The present invention further comprises 6 CDRs of anti-FXI antibodies of the αFXI-18611p family or αFXI-18611 family, or 1, 2 or 3 amino acid substitutions, additions, deletions or combinations thereof in which one or more of the CDRs. A first heavy / light chain pair of antibodies comprising at least that embodiment and six CDRs of the αFXI-18623p family, or one or more of the CDRs having one or more amino acid substitutions, additions, deletions or deletions. Provided are an anti-FXI antibody having a second layer / light chain pair of antibodies comprising at least the embodiment having a combination thereof and an antigen binding fragment thereof.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided is an anti-FXI diabodies in which one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof.

αFXI-18611p family, αFXI-18611 family or αFXI
Antibodies of the -18623p family of anti-FXI antibodies comprising at least 6 CDRs, or embodiments thereof in which one or more of the CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof. It can be modified in some way, in this case
That is, if the activity is expressed on a molar basis (parent antibody, ie each αFXI-1).
8611p family, αFXI-18611 family or αFXI-18623p
It possesses at least 10% of its FXI-binding activity (compared to family antibodies). Preferably, the antibody or antigen binding fragment of the invention is at least 20%, 50%, 70%, 80%, 90%, 95% or 10 of the FXI binding affinity as a parent antibody.
Own 0% or more. In addition, the antibody or antigen-binding fragment of the present invention is
It is intended that it may contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conserving variants" of the antibody) that do not substantially alter its biological activity.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
An isolated anti-FXI antibody, and an antigen-binding fragment thereof, wherein one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof. , And methods of using them, and also isolated comprising an isolated polypeptide immunoglobulin chain thereof, as well as an isolated polynucleotide encoding such a polypeptide, and such a polynucleotide. Vectors are provided.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Monoclonal anti-FXI antibodies, and antigen-binding fragments thereof, wherein one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof, and a plurality. Provided is a monoclonal composition containing the isolated monoclonal antibody of the above.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Provided are anti-FXI chimeric antibodies, wherein one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof, and methods of use thereof.

The present invention further relates to 6 CDRs, or 6 CDRs, of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
An anti-FXI fully human antibody, and an antigen-binding fragment thereof, wherein one or more of the six CDRs comprises at least one embodiment thereof having one, two or three amino acid substitutions, additions, deletions or combinations thereof. Including how to use them. In one embodiment of the invention, the fully human anti-FXI antibody or antigen-binding fragment thereof is a transgenic animal genetically modified to carry the fully human immunoglobulin gene, such as a mouse (eg, HUMAB mouse, etc.). For example, US Pat. Nos. 5,545,806, 5,569.
, 825, 5,625,126, 5,633,425, 5,661,016
No. 5,770,429, No. 5,789,650, No. 5,814,318, No. 5
, 874,299 and 5,877,397, and Harding et al., (19)
95) Ann. NY Acad. Sci. See 764: 536 546; or XENOMOUSE, eg, Green et al., 1999, J. Mol. Immunol. Met
An isolated product from (see hods 231: 11-23), or an isolated product from a phage or virus that expresses an immunoglobulin chain of an anti-FXI fully human antibody or antigen-binding fragment thereof.

In some embodiments, various constant domains can be linked to the _{CDR-derived VL} and _{VH regions provided in the present invention.} For example, heavy chain constant domains other than human IgG1 can be used, or hybrid IgG1 / I, where the individual intended use of the antibodies (or fragments) of the invention seeks alteration of effector function.
gG4 can be used.

Human IgG1 antibodies provide long half-lives and effector functions such as complement activation and antibody-dependent cellular cytotoxicity, which may not be desirable for all uses of the antibody. In such cases, for example, human IgG4 constant domain can be used. The present invention includes anti-FXI antibodies and antigen-binding fragments thereof comprising an IgG4 constant domain, such as antagonist human anti-FXI antibodies and fragments, and methods of their use. In one embodiment, the IgG4 constant domain is 2 in the EU system.
It can differ from the native human IgG4 constant domain (Swiss-Prot accession number P0186.11) at position 28 and at position corresponding to position 241 in the KABAT system, in which case it interferes with proper intrachain disulfide bond formation. Cysteine at position 106 (Cys106) and cysteine at position 109 (Cys109) (Cys226 and Cys229 in the EU system and Cys239 and C in the KABAT system)
Natural serine (Ser108) at position 108 of the HC constant domain is replaced with proline (Pro) to prevent potential interchain disulfide bonds (corresponding to position ys242). A
ngal et al., Mol. Immunol. See 30:105 (1993). Sch
urman et al., Mol. Immunol. 38: 1-8, (2001); SEQ ID NO: 14
See also 41. In other cases, modified IgG1 constant domains may be used to reduce effector function. For example, to significantly reduce ADCC and CDC, IgG1 isotypes may include substitutions of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 (Armour et al., Eu).
r J Immunol. 29 (8): 2613-24 (1999); Shiels et al., J Biol Chem. 276 (9): 6591-604 (2001)). In another embodiment, IgG HC is genetically modified to lack N-glycosylation of the asparagine (Asn) residue near position 297. The N-glycosylation consensus sequence is Asn-Xaa-Ser / Thr (where Xaa is any amino acid other than Pro), and in IgG1, the N-glycosylation consensus sequence is Asn-Se.
It is r-Thr. The modification can be accomplished by substituting the Asn codon at position 297 in the nucleic acid molecule encoding HC with another amino acid, such as the Gln codon. Alternatively, the Ser codon can be replaced with the Pro codon and the Thr codon can be replaced with S.
It can be replaced with any codon other than the codon of er. Such modified IgG1
The molecule has little or no detectable effector function. Alternatively, all three codons are modified.

In one embodiment of the invention, the αFXI-18611p family, αFXI-
Six CDRs of anti-FXI antibodies of the 18611 family or αFXI-18623p family, or one or more of the above six CDRs having one, two or three amino acid substitutions, additions, deletions or combinations thereof. The anti-FXI antibody containing at least the morphology is 2
Includes a complete tetrameric structure with two light chains and two heavy chains (including constant regions). The variable regions of each light / heavy chain pair form the antibody binding site. Therefore, in general, intact antibodies have two binding sites. Except for bispecific antibodies, their two binding sites are generally the same.

In certain embodiments, the present invention provides the anti-FXI antibodies shown in Table 1.

Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) described in Example 3 shows that the anti-FXI antibody containing HC and LC CDR is on the Apple 3 domain containing SEQ ID NO: 82 and SEQ ID NO: 83. It was shown to bind to a specific epitope of.

Therefore, the antibodies disclosed herein bind to the Apple 3 domain of FXI and
It inhibits FXI activation by FXIIa and also acts as an allosteric competing inhibitor of FXI activation by FXIa. The result of epitope mapping is that the "footprint" of the αFXI-18623p family on Apple 3 is F in FXIa.
It suggests that it overlaps with the IX-binding exosite.

Pharmaceutical Compositions and Administration In order to produce a pharmaceutical composition or sterile composition of an anti-FXI antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient. To do. For example, Remington's Pharmaceutical
Sciences and U.S.A. S. Pharmacopoeia: National Fo
rmulary, Mack Publishing Company, Easton, P
A (1984), and U.S.A. S. Physical Convention (Pharmacopeial Convention (
USP) 12601 Twinbrook Parkway, Rockville, MD
See 20852-1790, Continuous Updates on the Internet by USA.

Formulations of therapeutic and diagnostic substances can be made, for example, in the form of lyophilized powders, slurries, aqueous solutions or suspensions by mixing with acceptable carriers, excipients or stabilizers (eg Hardman). Et al. (2001) Goodman and Gilman's
The Pharmacological Bases of Therapeutic
s, McGraw-Hill, New York, NY; Gennaro (2000) R
emington: The Science and Practice of Pha
rmacy, Lippincott, Williams and Wilkins, New
York, NY; Avis et al. (Ed.) (1993) Pharmaceutical Dos
age Forms: Parental Medicines, Marcel
Dekker, NY; Lieberman et al. (Ed.) (1990) Pharmaceuti
cal Dose Forms: Tablets, Marcel Dekker, N
Y; Lieberman et al. (Ed.) (1990) Pharmaceutical Dosa
get Forms: Disperse Systems, Marcel Dekker,
NY; Winer and Kotkoskie (2000) Excipient Tox
icity and Safety, Marcel Dekker, Inc. , New
See York, NY).

In another embodiment, the composition comprising the antibody or antigen-binding fragment disclosed herein is used in Physicians' Desk Reference 2.
017 (Thomson Healthcare; 75st edition (2002)
Administer to the subject according to (November 1, 2014)).

The method of administration can be various. The appropriate route of administration is preferably parenteral or subcutaneous. Other routes of administration may include oral, transmucosal, intradermal, direct intraventricular, intravenous, intranasal, inhalation, infusion or intraarterial.

In certain embodiments, the anti-FXI antibody or antigen-binding fragment thereof can be administered by an invasive route, eg, injection. In a further embodiment of the invention, anti-FX
I-antibodies or antigen-binding fragments thereof or pharmaceutical compositions thereof can be administered intravenously, subcutaneously, intra-arterial, or by inhalation, aerosol transport. Non-invasive pathways (eg, oral,
For example, administration by pills, capsules or tablets) is also within the scope of the present invention.

The composition can be administered in a medical device known in the art. For example, the pharmaceutical compositions of the present invention can be administered by injection using a hypodermic needle, including, for example, a prefilled syringe or automatic infusion device.

The pharmaceutical compositions disclosed herein are, for example, U.S. Pat. Nos. 6,620,135, 6,096,002, 5,399,163, 5,383,851, No. 5. , 31
Nos. 2,335, 5,064,413, 4,941,880, 4,790,82
Needleless, such as the device disclosed in No. 4 or No. 4,596,556.
ess) It can also be administered by a subcutaneous injection device.

The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include US Pat. No. 4,48.
7,603 (which discloses an implantable microinjection pump for dispensing medicine at a controlled rate), US Pat. No. 4,447,233 (which discloses the exact infusion rate). Discloses a drug infusion pump for transporting drugs in US Pat. No. 4,447,224 (US Pat. No. 4,447,224)
It discloses a variable flow rate implantable infusion device for continuous drug delivery), US Pat. No. 4,439,196 (which discloses a permeable drug delivery system with a multi-chamber compartment. ) Includes those disclosed in. Many other such implants, transport systems and modules are well known to those of skill in the art.

The dosing regimen depends on several factors, including serum or tissue turnover rate of the therapeutic antibody, severity of symptoms, immunogenicity of the therapeutic antibody, and accessibility of target cells in the biological matrix. .. Preferably, the dosing regimen carries sufficient therapeutic antibodies that minimize unwanted side effects while at the same time providing improved targeted morbidity. Therefore, the amount of biological material carried depends, in part, on the individual therapeutic antibody and the severity of the condition being treated. Guidelines are available in choosing the appropriate dose of therapeutic dose antibody (eg, for example).
Wawrzynczak (1996) Antibody Therapy, Bios S
Scientist Pub. Ltd, Oxfordshire, UK; Kresina
(Edit) (1991) Monoclonal Antibodies, Cytokines
and Arthritis, Marcel Dekker, New York, NY
Bach (ed.) (1993) Monoclonal Antibodies and
Peptide Therapy in Autoimmunity Diseases, M
arcel Decker, New York, NY; Baert et al., 2003, New
Engl. J. Med. 348: 601-608; Milgrom et al., 1999, Ne
w Engl. J. Med. 341: 1966-1973; Slamon et al., 2001,
New Engl. J. Med. 344: 783-792; Beniaminovitz
Et al., 2000, New Engl. J. Med. 342: 613-619; Ghosh et al., 2003, New Engl. J. Med. 348: 24-32; Lipsky et al., 2
000, New Engl. J. Med. 343: 1594-1602)
..

The dosing regimen is adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or as indicated by the urgency of the treatment situation. It is possible to increase. It is particularly advantageous to formulate the parenteral composition in dosage unit form for ease of administration and uniformity of dosage. As used herein, the dosage unit form means a physically separated unit suitable as a unit dose for a subject to be treated. Each unit comprises a predetermined amount of active compound calculated to obtain the desired therapeutic effect, along with the required pharmaceutical carrier. Details of the dosage unit form described herein are:
Determined by (a) the unique characteristics of the antibody or antibody-binding fragment and the individual therapeutic effect to be achieved, and (b) the discipline-specific constraints of compounding such active molecules for the treatment of susceptibility in an individual. , Directly influenced by them (eg, Yang et al. (20)
03) New Engl. J. Med. 349: 427-434; Herold et al. (20)
02) New Engl. J. Med. 346: 1692-1698; Liu et al. (199)
9) J. Neurol. Neurosurg. Psych. 67: 451-456; Po
rtielji et al. (2003) Cancer Immunol. Immunother.
52: 133-144).

Kit In addition, the anti-FXI antibody or antigen binding described herein, along with one or more additional components including, but not limited to, another therapeutic agent described herein. Kits are provided that include one or more components that include, but are not limited to, sex fragments. The antibody or fragment and / or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier in the pharmaceutical composition.

In one embodiment, the kit contains an anti-FXI antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof in one container (eg, sterile glass or plastic vial) and another therapeutic agent. Contain in one container (eg, sterile glass or plastic vial).

In another embodiment, the kit is an anti-FXI antibody of the invention combined with one or more therapeutic agents that are formulated together in a single common container, optionally in a pharmaceutical composition. Alternatively, the combination of the present invention is included, which comprises an antigen-binding fragment thereof or a pharmaceutical composition thereof.

If the kit comprises a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for performing such administration. For example, the kit may include one or more of the above subcutaneous needles or other injection devices. Thus, the invention includes a kit comprising an injection device and the anti-FXI antibody or antigen-binding fragment thereof, eg, where the injection device comprises the antibody or fragment, or the antibody or fragment is separate. Present in the container.

The kit may include a package insert containing information about the pharmaceutical composition and dosage form in the kit. In general, such information assists patients and physicians in the effective and safe use of encapsulated pharmaceutical compositions and dosage forms. For example, the following information regarding the combinations of the invention may be provided in the package insert (insert): pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, excesses. dosage,
Appropriate dosage and administration, supply method, appropriate storage conditions, reference materials, manufacturer / distributor information and patent information.

Methods for Producing Antibodies and Antigen-Binding Fragments thereof The anti-FXI antibody and fragments thereof disclosed herein can also be produced by recombinant methods. In this embodiment, the nucleic acid encoding the antibody molecule is inserted into a vector (plasmid or virus) and transfected or transformed into a host cell.
It can be expressed in the host cell and secreted from the host cell. There are several methods for producing recombinant antibodies known in the art.

Mammalian cell lines available as hosts for the expression of antibodies or fragments disclosed herein are well known in the art and are American Type Cul.
Includes a number of immortalized cell lines available from the true Collection (ATCC). These are, among other things, Chinese Hamster Ovary (CHO) cells, NSOs, SPs.
2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells, 3T3 cells, human fetal kidney 29
Includes 3 (HEK-293) cells and numerous other cell lines. A particularly preferred cell line is
It is selected by determining which cell line has the highest expression level. Other cell lines that can be used include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, filamentous fungal cells [eg, Trichoderma ree].
sei)] and yeast cells [eg, Saccharoms cerevisiae (Saccharom)
yces cerevisiae or Pichia past
oris)]. In certain embodiments, the host cell can be a prokaryotic host cell, such as E. coli.

When a recombinant expression vector containing a heavy chain or an antigen-binding portion or fragment thereof, a light chain and / or a nucleic acid molecule encoding the antigen-binding fragment thereof is introduced into a host cell, expression of the antibody in the host cell, Alternatively, the antibody is produced by culturing the host cell under conditions and for a period of time sufficient to allow the antibody to be secreted into the medium in which the host cell is cultured. The antibody can be recovered from the medium and further purified or processed to obtain the antibody of the present invention.

In certain embodiments, HC and LCC of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family.
Nucleic acid molecules encoding HC and LC, including at least DR, or one or more of the six CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or HC. And / or LC variable region framework is 0, 1, 2, 3, 4,
Transpose a host cell with an expression vector containing a nucleic acid molecule encoding HC and LC containing at least 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, including the embodiment thereof. Fact.

In certain embodiments, a nucleic acid molecule encoding HC, including at least the HC CDRs of anti-FXI antibodies of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family, or one or more of the six CDRs is 1, 2 Or have 3 amino acid substitutions, additions, deletions or combinations thereof, and / or the HC and / or LC variable region framework is 0, 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, comprising a first expression vector comprising a nucleic acid molecule encoding HC comprising at least the embodiment thereof, and αFXI-18611.
Nucleic acid molecules encoding LC containing at least the LC CDRs of anti-FXI antibodies of the p family, αFXI-18611 family or αFXI-18623p family, or one or more of the six CDRs being 1, 2 or 3 amino acid substitutions, additions , Deletions or combinations thereof, and / or HC and / or LC variable region framework is 0,
A second comprising a nucleic acid molecule encoding LC comprising at least one embodiment thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.
Transfect host cells with an expression vector.

In certain embodiments, HC and LC are expressed as fusion proteins, in which the N-terminus of HC and LC is fused to a leader sequence that facilitates the transport of the antibody via the secretory pathway. .. An example of a reader sequence that could be used is MSVPTQ.
VLGLLLWLLTDARC (SEQ ID NO: 14) or MEWSVVLFFLSVTT
GVHS (SEQ ID NO: 15) is included.

The HC of a typical antibody in the present invention is SEQ ID NO: 34, 36, 38, 40, 42, 44.
, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
It can be encoded by a nucleic acid molecule having the nucleotide sequence shown in 72, 74, 76, 78 or 80.

The LC of a typical antibody in the present invention can be encoded by a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 27 or 32.

The present invention further relates to SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48, 50, 5
2,54,56,58,60,62,64,66,68,70,72,74,76,78
Alternatively, a plasmid or viral vector containing a nucleic acid molecule having an amino acid sequence of 80 is provided. The present invention further describes a nucleic acid molecule encoding the HC of an anti-FXI antibody of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family, or one or more of six CDRs substituted with one, two or three amino acids. , Additions, deletions or combinations thereof, and / or HC and / or LC variable region frameworks of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Nucleic acid molecules encoding HC of its embodiment, including amino acid substitutions, additions, deletions or combinations thereof, and αF.
XI-18611p family, αFXI-18611 family or αFXI-18
Nucleic acid molecule encoding LC of 623p family of anti-FXI antibodies, or 6 CDs
One or more of R has 1, 2 or 3 amino acid substitutions, additions, deletions or combinations thereof, and / or HC and / or LC variable region frameworks are 0, 1, 2, 3,
Provided is a plasmid or viral vector containing 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof with a nucleic acid molecule encoding LC of the embodiment.

The present invention further comprises a plasmid or viral vector containing a nucleic acid molecule encoding the HC of an anti-FXI antibody of the αFXI-18611p family, αFXI-18611 family or αFXI-18623p family, and the αFXI-18611p family, α.
Provided are a plasmid or viral vector containing a nucleic acid molecule encoding the LC of an anti-FXI antibody of the FXI-18611 family or the αFXI-18623p family.

The present invention further relates to nucleic acid molecules encoding the HC of anti-FXI antibodies of the αFXI-18611p family, the αFXI-18611 family or the αFXI-18623p family.
Alternatively, one or more of the six CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or the HC and / or LC variable region framework is 0, 1, Nucleic acid molecules encoding HC of its embodiment, including 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, and αFXI-1.
8611p family, αFXI-18611 family or αFXI-18623p
A nucleic acid molecule encoding the LC of a family of anti-FXI antibodies, or one or more of the six CDRs having one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or HC. And / or LC variable region framework is 0, 1, 2, 3, 4, 5,
Provided is a host cell containing one or more plasmids or viral vectors containing a nucleic acid molecule encoding LC of the embodiment containing 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. To do. In certain embodiments, the host cell is a CHO or HEK-293 host cell.

Antibodies can be recovered from the medium using standard protein purification methods. In addition, expression of the antibody of the invention (or other portion thereof) from the producing cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions.

In general, glycoproteins produced in an individual cell line or transgenic animal have a glycosylation pattern characteristic of the glycoprotein produced in the cell line or transgenic animal (eg, Croset et al., J. Biotechnol.
See 161: 336-348 (2012)). Therefore, the individual glycosylation pattern of an antibody depends on the individual cell line or transgenic animal used to produce the antibody. However, it is encoded by the nucleic acid molecules provided in the present invention.
Alternatively, all antibodies comprising the amino acid sequence provided in the present invention constitute the present invention regardless of the glycosylation pattern that the antibody may have.

The following examples are intended to facilitate a further understanding of the present invention.

General Methods Standard methods in molecular biology are Sambook, Fritsch and Man.
iatis (1982 & 1989 2nd Edition, 2001 3rd E
division) Molecular Cloning, A Laboratory Ma
natural, Cold Spring Harbor Laboratory Press
, Cold Spring Harbor, NY; Sambook and Russel
l (2001) Molecular Cloning, 3rded, Cold Spri
ngHarbor Laboratory Press, Cold Spring H
arbor, NY; Wu (1993) Recombinant DNA, Vol. 217
, Academic Press, San Diego, CA. It is described in. The standard method is Ausbel et al. (2001) Current Protocols in
Molecular Biology, VoIs. 1-4, John Wiley an
d Sons, Inc. Also described in New York, NY, this includes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), complex sugar and protein expression (Vol. 1). 3),
Also, bioinformatics (Vol.4) is described.

Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization have been described (Coligan et al. (2000) Current Pr.
otocols in Protein Science, Vol. 1, John Wi
ray and Sons, Inc. , New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (eg, for example).
Coligan et al. (2000) Current Protocols in Prote
in Science, Vol. 2, John Wiley and Sons, Inc
.. , New York; Ausubel et al. (2001) Current Protoco
ls in Molecular Biology, Vol. 3, John Wiley
and Sons, Inc. , NY, NY, pp. 16.0.5-16.22.17;
Sigma-Aldrich, Co., Ltd. (2001) Products for Life
Science Research, St. Louis, MO; pp. 45-89; A
mersham Pharmacia Biotech (2001) BioDirect
ory, Piscataway, N. et al. J. , Pp. See 384-391). Production, purification and fragmentation of polyclonal and monoclonal antibodies have been described (Colligan et al. (2001) Current Protocols in Imm).
unology, Vol. 1, John Wiley and Sons, Inc. , N
ew York; Harlow and Lane (1999) Using Antibody
ies, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY; Harlow and Lane, supra).
Standard techniques for characterizing ligand / receptor interactions are available (eg, for example.
Coligan et al. (2001) Current Protocols in Immuno
logy, Vol. 4, John Wiley, Inc. , New York).

Monoclonal antibodies, polyclonal antibodies and humanized antibodies can be produced (eg, Sheperd and Dean (eds.) (2000) Monoclonal Antib.
ideas, Oxford Univ. Press, New York, NY; Kont
ermann and Dubel (eds.) (2001) Antibody Engineer
ing, Springer-Verlag, New York; Harlow and La
ne (1988) Antibodies A Laboratory Manual, C
old Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, pp. 139-243; Carpenter et al. (2)
000) J. Immunol. 165: 6205; He et al. (1998) J. et al. Immuno
l. 160: 1029; Tang et al. (1999) J. Mol. Biol. Chem. 274: 27
371-27378; Baca et al. (1997) J. Mol. Biol. Chem. 272: 106
78-10684; Chothia et al. (1989) Nature 342: 877-88
3; Foote and Winter (1992) J. et al. Mol. Biol. 224: 487
-499; see US Pat. No. 6,329,511).

An alternative to humanization is to use the human antibody library presented on the phage, or the human antibody library in transgenic mice (Vaugha).
n et al. (1996) Nature Biotechnology. 14: 309-314; Bar
bass (1995) Nature Medicine 1: 837-839; Mende
z et al. (1997) Nature Genetics 15: 146-156; Hooge
nboom and Chames (2000) Immunol. Today 21: 371
-377; Barbas et al. (2001) Page Display: A Labora
tory Manual, Cold Spring Harbor Laborator
y Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Prot
eins: A Laboratory Manual, Academic Press,
San Diego, CA; de Bruin et al. (1999) Nature Biote
chnor. 17: 397-399).

Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other purposes and include, for example, antibodies bound to dyes, radioisotopes, enzymes or metals such as colloidal gold (
For example, Le Doussal et al. (1991) J. Mol. Immunol. 146: 169-1
75; Gibellini et al. (1998) J. Mol. Immunol. 160: 3891-38
98; Hsing and Bishop (1999) J. Mol. Immunol. 162: 280
4-2811; Everts et al. (2002) J. Mol. Immunol. 168: 883-88
9).

Methods for flow cytometry including a fluorescently labeled cell preparative detection system (FACS) are available (eg, Owens et al. (1994) Flow Cytometry Pri.
nciples for Clinical Laboratory Laboratory
, John Wiley and Sons, Hoboken, NJ; Givan (20)
01) Flow Cytometry, 2nd ed. Willey-Liss, Hob
oken, NJ; Shapiro (2003) Practical Flow Cyto
See meter, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, polypeptides and antibodies, including, for example, nucleic acid primers and probes, for use as diagnostic reagents are available (Mol).
ecular Probes (2003) Catalog, Molecule P
robes, Inc. , Eugene, OR; Sigma-Aldrich (2003)
Catalog, St. Louis, MO).

Standard histological methods of the immune system are described (eg, Muller-Harme).
link (ed.) (1986) Human Symus: Histopathology
and Pathology, Springer Verlag, New York,
NY; Hitt et al. (2000) Color Atlas of Histology,
Lippincott, Williams, and Wilkins, Phila, PA
Louis et al. (2002) Basic Histology: Text and At
See las, McGraw-Hill, New York, NY).

For example, software packages and databases for determining antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites and sequence alignments are available (eg, GenBank, Vector NTI).
(Registered Trademark) Suite (Informax, Inc, Bethesda, MD); GC
G Wisconsin Package (Accelrys, Inc., San Di)
ego, CA); DeCypher® (TimeLogic Corp., C)
rhythm Bay, Nevada); Menne et al. (2000) Bioinform
atics 16: 741-742; Menne et al. (2000) Bioinformat
ics Applications Note 16: 741-742; Wren et al. (2)
002) Complete. Methods Programs Biomed. 68:17
7-181; von Heijne (1983) Euro. J. Biochem. 133:
17-21; von Heijne (1986) Nucleic Acids Res.
14: 4683-4690).

Human FXI and FIX thymogen are Haematologic Technology
ies, Inc. Available from Essex Junction, VT, high molecular weight (
HMW) Kininogen is Enzyme Research Laboratories,
Available from South Bend, IN, ellagic acid is Pacific Hemo
It is available from stasis, Thermo Fisher, Waltham, and MA.

1A and 1B show the coagulation cascade, FXI, FXI mAb and four new oral anticoagulants (NOACs). FIG. 1A is a diagram showing FXI in a coagulation cascade (consisting of an endogenous pathway and an exogenous pathway). FXI-targeted mAbs can result in functional neutralization by blocking FXI activation by XIIa and / or thrombin, or FXIa activity against FIX. Antibodies in the present invention can provide double blockade of FXIa-mediated activation of FIX and at least FXIIa-mediated conversion of FXI to FXIa. Four NOACs (rivaloxaban, apixaban, edoxaban, dabigatran) targeting either FXa or thrombin have been shown. 1A and 1B show the coagulation cascade, FXI, FXI mAb and four new oral anticoagulants (NOACs). FIG. 1B shows the domain structure of FXI. FXI is a dimer composed of the same 80 kDa subunit, with each subunit starting at the N-terminus consisting of four apple domains (1, 2, 3 and 4) and a catalytic domain (CAT). The antibodies disclosed herein bind to the Apple 3 domain. FIG. 2 shows the structure of factor XI and the Apple 3 domain, identifying peptides protected from deuterium by the αFXI-18611 and αFXI-18623p family anti-FXI antibodies. 184 residues of arginine, which is a critical residue in the FIX binding exosite, are shown. Peptides in the Apple 3 domain that show no difference in deuteration are shown in light gray. Peptides for which no data were available are colored dark gray. The catalytic domain is not shown. 3A and 3B show the FXI amino acid residue bound by the anti-FXI antibody αFXI-18611 IgG4 HC (S228P) (E1) (L105) / LC kappa and αFXI-18623p IgG4 HC (S228P) (Q1) / LC kappa, respectively. The deuterium-labeled difference heatmap of the group is shown. 3A and 3B show the FXI amino acid residue bound by the anti-FXI antibody αFXI-18611 IgG4 HC (S228P) (E1) (L105) / LC kappa and αFXI-18623p IgG4 HC (S228P) (Q1) / LC kappa, respectively. The deuterium-labeled difference heatmap of the group is shown. 4A, 4B and 4C show the amino acid sequences of the HC and LC domains of the αFXI 18611p and αFXI 18611 family antibodies. Heavy and light chain CDRs have been identified as HC-CDR1, HC-CDR-2, HC-CDR3, LC-CDR1, LC-CDR2 and LC-CDR3, respectively. 4A, 4B and 4C show the amino acid sequences of the HC and LC domains of the αFXI 18611p and αFXI 18611 family antibodies. Heavy and light chain CDRs have been identified as HC-CDR1, HC-CDR-2, HC-CDR3, LC-CDR1, LC-CDR2 and LC-CDR3, respectively. 4A, 4B and 4C show the amino acid sequences of the HC and LC domains of the αFXI 18611p and αFXI 18611 family antibodies. Heavy and light chain CDRs have been identified as HC-CDR1, HC-CDR-2, HC-CDR3, LC-CDR1, LC-CDR2 and LC-CDR3, respectively. 5A and 5B show the amino acid sequences of the HC and LC domains of the αFXI 18623p family antibody. Heavy and light chain CDRs have been identified as HC-CDR1, HC-CDR-2, HC-CDR3, LC-CDR1, LC-CDR2 and LC-CDR3, respectively. 5A and 5B show the amino acid sequences of the HC and LC domains of the αFXI 18623p family antibody. Heavy and light chain CDRs have been identified as HC-CDR1, HC-CDR-2, HC-CDR3, LC-CDR1, LC-CDR2 and LC-CDR3, respectively. FIG. 6 shows the activated partial thromboplastin of αFXI-18611 IgG4 HC (S228P) (E1) (L105) / LC kappa (A) and αFXI-18623p IgG4 HC (S228P) (Q1) / LC kappa (B) in human plasma. The results of the time (aPTT) assay are shown, which are expressed as the rate of increase (%) relative to baseline. FIG. 7 shows the activated partial thromboplastin of αFXI-18611 IgG4 HC (S228P) (E1) (L105) / LC kappa (A) and αFXI-18623p IgG4 HC (S228P) (Q1) / LC kappa (B) in cynomolgus monkey plasma. The results of the time (aPTT) assay are shown, which are expressed as the rate of increase (%) relative to baseline. FIG. 8 shows the activated partial thromboplastin of αFXI-18611 IgG4 HC (S228P) (E1) (L105) / LC kappa (A) and αFXI-18623p IgG4 HC (S228P) (Q1) / LC kappa (B) in rhesus monkey plasma. The results of the time (aPTT) assay are shown, which are expressed as the rate of increase (%) relative to baseline. FIG. 9 shows a comparison of aPTT results for αFXI-18611 IgG4 HC (S228P) (E1) (L105) / LC kappa in human plasma, cynomolgus monkey and rhesus monkey plasma, which are expressed as percentages of increase relative to baseline. ing. FIG. 10 shows a comparison of aPTT results for αFXI-18623p IgG4 HC (S228P) (Q1) / LC kappa in human plasma, cynomolgus monkey and rhesus monkey plasma, which are expressed as percentages of increase relative to baseline. FIG. 11 shows a BIAcore sensorgram showing the kinetics of binding of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa to human, cynomolgus monkey and rhesus monkey FXI and other human and NHP coagulation cascade proteins. FIG. 12 shows the BIAcore sensorgram showing the kinetics of binding of αFXI-18623p IgG4 HC (S228P) (Q1) / LC kappa to human, cynomolgus monkey and rhesus monkey FXI and other human and NHP coagulation cascade proteins. FIG. 13 shows a schematic diagram of the cynomolgus monkey AV shunt test method. Intravenous bolus of vehicle or αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa (antibody) (0.01-1.0 mg / kg) in anesthetized monkeys pre-attached with femoral artery and venous catheters (Test substance administration). The AV shunt was inserted as described herein (insertion of the AV shunt). Blood was flushed via an AV shunt for 40 minutes. The contact between the silk thread suspended inside the tube and the blood formed a blood clot. Blood clots were weighed as described herein. Blood samples were obtained to measure antibody circulation levels, aPTT and PT (stars). 14A-D show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa (antibody) on AV shunt clot formation, aPTT and PT in cynomolgus monkey AV shunt model. FIG. 14A shows clot weight measured after two consecutive AV shunts in the same animal. The vehicle was administered to the animal in the first shunt (shunt # 1) and then the antibody (0.01-1.0 mg / kg IV) in the second shunt (shunt # 2) as shown. Was administered. Increasing the dose of antibody resulted in the formation of smaller clots. 14A-D show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa (antibody) on AV shunt clot formation, aPTT and PT in cynomolgus monkey AV shunt model. The rate of clot weight inhibition (FIG. 14B) and the rate of change in aPTT (FIG. 14C) increased with increasing plasma concentration of the antibody. In contrast, PT (FIG. 14D) remained relatively unchanged at all antibody concentrations. 14A-D show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa (antibody) on AV shunt clot formation, aPTT and PT in cynomolgus monkey AV shunt model. The rate of clot weight inhibition (FIG. 14B) and the rate of change in aPTT (FIG. 14C) increased with increasing plasma concentration of the antibody. In contrast, PT (FIG. 14D) remained relatively unchanged at all antibody concentrations. 14A-D show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa (antibody) on AV shunt clot formation, aPTT and PT in cynomolgus monkey AV shunt model. The rate of clot weight inhibition (FIG. 14B) and the rate of change in aPTT (FIG. 14C) increased with increasing plasma concentration of the antibody. In contrast, PT (FIG. 14D) remained relatively unchanged at all antibody concentrations. FIG. 15 shows a schematic diagram of the template bleeding time method for cynomolgus monkeys. Template bleeding times in the buccal mucosa (inner lip), finger toes and distal tail in anesthetized cynomolgus monkeys at baseline (before treatment) and treatment # 1 (vehicle) and treatment # 2 (vehicle or αFXI-18623p IgG4) Measured after administration of HC (S228P) (E1) / LC kappa, 10 mg / kg IV). Blood samples for measuring circulating levels, aPTT and PT of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa were taken as shown. 16A-F show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa on template bleeding time measured in cynomolgus monkeys. Template bleeding time was measured in the buccal mucosa (FIGS. 16A, 16D), finger toes (FIGS. 16B, 16E) and distal tail (FIGS. 16C, 16F). Vehicle-vehicle as process 1 and process 2 in study session 1 and vehicle-αFXI-18623p IgG4 HC (S228P) (E1) / LC as process 1 and 2 in study session 2 using one-sided student's t-test. For kappa, the treatment effect on bleeding time (αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa pair by comparing the absolute bleeding time (left panel) and the rate of change in bleeding time (%) (right panel). Vehicle) was evaluated. 16A-F show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa on template bleeding time measured in cynomolgus monkeys. Template bleeding time was measured in the buccal mucosa (FIGS. 16A, 16D), finger toes (FIGS. 16B, 16E) and distal tail (FIGS. 16C, 16F). Vehicle-vehicle as process 1 and process 2 in study session 1 and vehicle-αFXI-18623p IgG4 HC (S228P) (E1) / LC as process 1 and 2 in study session 2 using one-sided student's t-test. For kappa, the treatment effect on bleeding time (αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa pair by comparing the absolute bleeding time (left panel) and the rate of change in bleeding time (%) (right panel). Vehicle) was evaluated. 16A-F show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa on template bleeding time measured in cynomolgus monkeys. Template bleeding time was measured in the buccal mucosa (FIGS. 16A, 16D), finger toes (FIGS. 16B, 16E) and distal tail (FIGS. 16C, 16F). Vehicle-vehicle as process 1 and process 2 in study session 1 and vehicle-αFXI-18623p IgG4 HC (S228P) (E1) / LC as process 1 and 2 in study session 2 using one-sided student's t-test. For kappa, the treatment effect on bleeding time (αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa pair by comparing the absolute bleeding time (left panel) and the rate of change in bleeding time (%) (right panel). Vehicle) was evaluated. 16A-F show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa on template bleeding time measured in cynomolgus monkeys. Template bleeding time was measured in the buccal mucosa (FIGS. 16A, 16D), finger toes (FIGS. 16B, 16E) and distal tail (FIGS. 16C, 16F). Vehicle-vehicle as process 1 and process 2 in study session 1 and vehicle-αFXI-18623p IgG4 HC (S228P) (E1) / LC as process 1 and 2 in study session 2 using one-sided student's t-test. For kappa, the treatment effect on bleeding time (αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa pair by comparing the absolute bleeding time (left panel) and the rate of change in bleeding time (%) (right panel). Vehicle) was evaluated. 16A-F show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa on template bleeding time measured in cynomolgus monkeys. Template bleeding time was measured in the buccal mucosa (FIGS. 16A, 16D), finger toes (FIGS. 16B, 16E) and distal tail (FIGS. 16C, 16F). Vehicle-vehicle as process 1 and process 2 in study session 1 and vehicle-αFXI-18623p IgG4 HC (S228P) (E1) / LC as process 1 and 2 in study session 2 using one-sided student's t-test. For kappa, the treatment effect on bleeding time (αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa pair by comparing the absolute bleeding time (left panel) and the rate of change in bleeding time (%) (right panel). Vehicle) was evaluated. 16A-F show the effect of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa on template bleeding time measured in cynomolgus monkeys. Template bleeding time was measured in the buccal mucosa (FIGS. 16A, 16D), finger toes (FIGS. 16B, 16E) and distal tail (FIGS. 16C, 16F). Vehicle-vehicle as process 1 and process 2 in study session 1 and vehicle-αFXI-18623p IgG4 HC (S228P) (E1) / LC as process 1 and 2 in study session 2 using one-sided student's t-test. For kappa, the treatment effect on bleeding time (αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa pair by comparing the absolute bleeding time (left panel) and the rate of change in bleeding time (%) (right panel). Vehicle) was evaluated. FIG. 17A shows the concentration-time profile after αFXI-18623p IgG4 HC (S228P) (E1) / LC Kappa IV administration in rhesus monkeys. The plasma concentration-time profile of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa in rhesus monkeys is shown. There were 4 animals in each dose group. Each line represents the average for each group. FIG. 17B shows the aPTT-time profile in rhesus monkeys. An aPTT-time profile for αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa is shown for each dose group. There were 4 animals in each dose group. Each symbol represents an individual animal's aPTT time profile at each time point. Each line represents the average for each group.

Example 1
In this example, the anti-FXI antibody αFXI-18611 IgG4 HC (S228)
P) (E1) (L105) / LC Kappa and αFXI-18623p IgG4 HC
The binding kinetics of (S228P) (Q1) / LC kappa and either human FXI thymogen or non-human primate (NHP) FXI timogen was measured using the following assay.

Human FXI and FXIa Binding Dynamics Assay Protocol The binding kinetics and affinity of protein-protein interactions between anti-FXI antibodies and human FXI thymogens or FXIa is based substantially on SPR (Surface Plasmon Resonance) as follows: ProteinOn XPR36 (Bi), an optical biosensor
It was measured using o-Rad).

GLC low density sensor chips were washed with 0.5% sodium dodecyl sulfate, 50 mM sodium hydroxide and 100 mM hydrochloric acid at a flow rate of 30 μL / sec for 60 seconds across all vertical and horizontal channels. The alginate chip surfaces of all six vertical channels (L1 to L6) were then activated at 1 × EDC / sNHS at a flow rate of 30 μL / sec for 150 seconds.
A mouse Fc-directed anti-human IgG polyclonal antibody (capture antibody) diluted to 1.25 μg / mL in 10 mM sodium acetate (pH 5.0) was then applied over all six vertical channels at a flow rate of 25 μL / sec to 300. Injecting for seconds, amine coupling to endogenous lysine bound about 300 response units (RU) of capture antibody per channel to the surface of the activated chip. 1M ethanolamine HCl was then injected over all six vertical channels to neutralize the remaining reactive surface amines. Then, to achieve a saturation capture level of about 80 RU, capture antibody (at a concentration of 5 μg / mL in 10 mM sodium acetate (pH 5.0)).
Anti-FX in a separate vertical flow path coated with L2, L3, L4, L5 or L6)
Each I antibody was injected at 25 μL / min for 60 seconds. 10 mM sodium acetate (pH 5.0) (buffer only) was injected into the vertical channel L1 as a reference control.

After capture of anti-FXI antibody, running buffer (1 x HBS-N, 5 mM CaCl)
₂ , 0.005% P20, pH 7.4) is injected into all horizontal channels (A1 to A6) for 5 minutes and dissociated at 25 μL / min for 20 minutes to allow non-specific binding anti-FXI antibody from the chip surface. Removed. Then, the on-rate of human FXI or FXa against the capture anti-FXI antibody (o).
n-rate) _{(k a)} in order to measure, diluted with 6 point titration of human FXI or FXIa (running buffer 0,0.25,0.5,1.0,2.0,4. 0nM)
Was injected horizontally for 8 minutes across all 6 vertical channels. The bound zymogen was then dissociated in running buffer at 25 μL / min for 60 minutes to off-rat.
e) (k _d ) was measured. Binding kinetics and affinity _{(K D),} it was determined using specific software (Bio-Rad) in the apparatus. They are shown in Table 2.

Non-human primate FXI thymogen / FXIa binding kinetics assay protocol Anti-FXI antibody and non-human primate (NHP; crab and rhesus monkey) FXI thymogen or protein-protein interaction binding kinetics and affinity, SPR ( Protein, an optical biosensor based on surface plasmon resonance)
Measurements were made using eOn XPR36 (Bio-Rad).

GLC low density sensor chips were washed with 0.5% sodium dodecyl sulfate, 50 mM sodium hydroxide and 100 mM hydrochloric acid at a flow rate of 30 μL / sec for 60 seconds across all vertical and horizontal channels. The alginate chip surfaces of all six vertical channels (L1 to L6) were then activated at 1 × EDC / sNHS at a flow rate of 30 μL / sec for 150 seconds.
Then, mouse Fc-directed anti-human IgG polyclonal antibody (capture antibody) diluted to 30 μg / mL in 10 mM sodium acetate (pH 5.0) was injected over all six vertical channels at a flow rate of 25 μL / sec for 150 seconds. Then, by amine coupling to endogenous lysine, saturated binding of about 4500 response units (RU) of the capture antibody per channel to the surface of the activated chip was achieved. 1M ethanolamine HCl was then injected over all six vertical channels to neutralize the remaining reactive surface amines. Then running buffer (1 x HBS-N, 5 mM CaCl ₂ , 0.0) to achieve a saturation capture level of about 40 RU.
At a concentration of 0.415 μg / mL in 05% P20, pH 7.4), capture antibody (L2, L)
3. Anti-FXI antibodies were injected at 25 μL / min for 60 seconds, respectively, into separate vertical channels coated with L4, L5 or L6). Only running buffer was injected into the vertical flow path L1 as a reference control. After capture of the anti-FXI antibody, running buffer was injected into all horizontal channels (A1-A6) for 5 minutes and dissociated at 25 μL / min for 20 minutes to remove non-specifically bound anti-FXI antibody from the chip surface. .. Then, NHP for capture anti-FXI antibody
To measure the FXI on-rate _{(on-rate) (k a} ), 6 point titration NH
P FXI or FXIa (0,0.25,0.5 diluted in running buffer)
, 1.0, 2.0, 4.0 nM) was injected horizontally over all six vertical channels for 8 minutes. Then bound FXI zymogen or dissociated for 60 min at 25 [mu] L / min in running buffer to FXIa, was measured off-rate _{(off-rate) (k d} ). Binding kinetics and affinity _{(K D),} it was determined using specific software (Bio-Rad) in the apparatus. They are shown in Table 2.

Example 2
Effect of anti-FXI antibody on FXI to FXIa activation by FXIIa in the presence of high molecular weight (HMW) kininogen and ellagic acid Anti-FXI antibody αFXI-18611 IgG4 HC on FXI thymogen activation
(S228P) (E1) (L105) / LC Kappa and αFXI-18623p Ig
To measure the effect of G4 HC (S228P) (Q1) / LC kappa, the antibody is FXI active using a conjugated enzyme assay that measures FXIa-mediated proteolysis of tripeptide fluorophore (GPR-AFC). It is possible to determine whether to inhibit the conversion itself. For these experiments, anti-FXI antibody is pre-incubated with FXI thymogen for 1 hour. Activation of FXI into FXIa is induced by the addition of FXIIa in the presence of HMW kininogen and ellagic acid. Then FXI for the tripeptide fluorophore substrate
a Catalytic activity is measured as a reading result of tymogen activation. The conjugated assay is also performed in the absence of HMW kininogen as a control. Anti-FXI antibody in 11-point dose titration starting from a concentration of 1 μM in a 3-fold dilution series in a Corning 3575 unbound surface microplate, 50 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂
, 0.1% PEG-8000 (pH 7.4) in human FXI (Haematologi)
c Technologies, Inc. , Cat # HCXI-0150, final concentration 30 nM) and HMW kininogen (Enzyme Research Labora)
Pre-incubated at 25 ° C. for 2 hours with tories, Cat # HK, final concentration 280 nM). Next, ellagic acid-containing Pacific Hemostasis APT
T-XL Reagent (Thermo Scientific, Cat # 100403,10
0 μM stock concentration, final concentration 2 μM) and freshly diluted coagulation factor XIIa (E)
nzyme Research Laboratories, Cat # HFXIIa
, Final concentration 50 pM) was added to initiate the activation reaction. The reaction was allowed to proceed at 25 ° C. for 1 hour, at which time 1 μM corn trypsin inhibitor (Haematologi).
c Technologies, Inc. , Cat # CTI-01)
Quenched it. Z-GPR by continuously monitoring fluorescence at 400/505 nm for 10 minutes using a Tecan Infinity M200 plate reader.
The newly activated FXIa enzyme activity was detected by measuring the rate of cleavage of the -AFC substrate (Sigma, Cat # C0980-10MG, final concentration 150 μM). The inhibition rate (%) for each data point is recalculated from the RFU / min data and GraphPad Pr
Analysis was performed using the ism software using a four-parameter equation of log (inhibitor) vs. response. The results are shown in Table 3.

From FXI to FXIa by FXIIa in the absence of HMW kininogen and ellagic acid
Effect of anti-FXI antibody on activation of the anti-FXI antibody of the present invention in 11-point dose titration starting from a concentration of 1 μM in a 3-fold dilution series.
In Corning 3575 unbound surface microplate, 50 mM HEPES, 1
50 mM NaCl, 5 mM CaCl ₂ , 0.1% PEG-8000 (pH 7.4)
Medium, Human FXI (Humanotropic Technologies, Inc., C.
Pre-incubated at 25 ° C. for 2 hours with at # HCXI-0150, final concentration 30 nM). Then, the newly diluted coagulation factor XIIa (Enzyme Leather)
The activation reaction was initiated by the addition of ch Laboratories, Cat # HFXIIa, final concentration 15 nM). The reaction was allowed to proceed at 25 ° C. for 1 hour, at which time 1 μ.
M Corn Trypsin Inhibitor (Haematologic Technology)
es, Inc. , Cat # CTI-01), quenching it. Te
Z-GPR-AFC substrate (Sigma) by continuously monitoring fluorescence at 400/505 nm for 10 minutes using a can Infinite M200 plate reader.
, Cat # C0980-10MG, final concentration 150 μM) was measured to detect newly activated FXIa enzyme activity. Inhibition rate (%) for each data point
) Is recalculated from the RFU / minute data, and with GraphPad Prism software, l
Analysis was performed using a four-parameter formula of og (inhibitor) vs. response. The results are shown in Table 3.

Taken together, these mechanistic studies have shown that these anti-FXI antibodies functionally neutralize FXI by interfering with FXIa activation by FXIIa and by inhibiting FXIa catalytic activity on natural substrates. ing.

Example 3
Epitope mapping of anti-FXI antibody by hydrogen-deuterium exchange mass spectrometry αFXI-18611 IgG4 HC (S228P) (E1) (E1) for human FXI
L105) / LC Kappa and αFXI-18623p-IgG4 (S228P) (Q1)
) / LC kappa contact regions were determined using hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS measures the uptake of deuterium into the amide skeleton of proteins, and changes in this uptake are affected by hydrogen solvent exposure. Deuterium exchange levels were compared between the antigen-only sample and the antibody-bound sample to identify the antigen region that could come into contact with the antibody. Human factor XI has the amino acid sequence set forth in SEQ ID NO: 81. The dimer factor XI was pre-incubated with the antibody and then incubated in deuterium buffer. The uptake of deuterium into factor XI was measured by mass spectrometry.

The human factor XI region protected from deuteration by the antibody is epitope-A IFFNT
VF (residues 185 to 192 of factor XI; SEQ ID NO: 82) and epitope-B PSTR
IKKSKALSG (residues 247-259 of factor XI; SEQ ID NO: 83). 3A and 3B show the antibody αFXI-18611 IgG4 HC (S228P) (E, respectively).
1) (L105) / LC Kappa and αFXI-18623p IgG4 HC (S22)
8P) (Q1) / Shows a deuterium-labeled difference heatmap of factor XI amino acid residues bound by LC kappa. These amino acid sequences are located on the Apple 3 domain of factor XI (
Figure 3). No significant deuteration changes were observed in Apple 1, 2, 4 or the catalytic domain. This indicates that they are not involved in αFXI-18623 binding. Thus, the epitopes recognized by αFXI-18623p-IgG4 (S228P) / kappa include epitopes A and B.

Example 4
FIX is an endogenous protein substrate for FXIa, the active protease of FXI thymogen. FXIa activates FIX into FIXa, perpetuating the coagulation cascade. F
Inhibition of FXIa-mediated activation of IX is one possible mechanism of action (MOA) for FXI mAbs. FX using full length FIX zymogen to examine this MOA
The Ia enzyme assay was developed.

FXIa Protease Activity Against Small Tripeptide Substrates Anti-FXI antibody 50 m in Corning 3575 unbound surface microplate
M HEPES, 150 mM NaCl, 5 mM CaCl ₂ , 0.1% PEG-80
In 00 (pH 7.4), human FXIa (Sekisui Diagnostics, Ex)
Pre-incubated at 25 ° C. for 2 hours with ton, PA, Cat # 4011A, final concentration 100 pM). Z-GP by continuously monitoring fluorescence at 400/505 nm for 10 minutes using a Tecan Infinity M200 plate reader.
The newly activated FXIa enzyme activity was determined by measuring the rate of cleavage of the R-AFC substrate (Sigma, Cat # C0980-10MG, final concentration 100 μM). 3
The final concentration of the antibody in an 11-point dose titration starting at 1 μM in a double dilution series. Inhibition rates (%) for each data point were recalculated from RFU / min data and analyzed with GraphPad Prism software using a four-parameter formula of log (inhibitor) vs. response. The results are shown in Table 4.

FIX to FIXa Activation by FIXI FIX is an endogenous protein substrate for FIXIa, the active protease of FXI thymogen. FXIa activates FIX into FIXa, perpetuating the coagulation cascade. F
Inhibition of FXIa-mediated activation of IX is one possible mechanism of action (MOA) for FXI mAbs. To investigate this MOA, we developed the FXIa enzyme assay using a full-length FIX.

An 11-point dose titration of anti-FXI antibody starting at a concentration of 1 μM in a 3-fold dilution series, Corn.
ing 3575 In a non-bound surface microplate, 50 mM HEPES, 150 mM
Preincubated with human FXIa (Sekisui Diagnostics, Cat # 4011A, final concentration 100 pM) in NaCl, 5 mM CaCl _{2, 0.1% PEG-8000 (pH 7.4) for 2 hours at 25 ° C.} Then FIX (Heme)
Atotropic Technologies, Inc. , Cat # HCIX-00
The activation reaction was initiated by the addition of 40-C, final concentration 300 nM) and allowed to proceed at 25 ° C. for 1 hour, at which time the anti-FXI antibody (WO201316) against the catalytic site of the light chain of FXI.
It was quenched by the addition of 100 nM of anti-FXI antibody 076D-M007-H04) disclosed in 7669. Cyclohexyl-GGR-AFC substrate (CPC Scientific, Cat # 83) by continuously monitoring fluorescence at 400/505 nm for 10 minutes using a Tecan Infinity M200 plate reader.
The newly activated FIXa enzyme activity was detected by measuring the rate of cleavage (9493, final concentration 300 μM). Inhibition rates (%) for each data point were recalculated from RFU / min data and analyzed with GraphPad Prism software using a four-parameter formula of log (inhibitor) vs. response. The results are shown in Table 4.

As shown in Table 4, in enzyme assays using synthetic tripeptide fluorophore substrates, the antibodies did not inhibit FXIa catalytic activity, whereas both antibodies were potent in assays using natural full length substrates. It was an inhibitor. This data is consistent with antibodies that act as allosteric competing inhibitors of FIX activation by FXIa.
This is consistent with the results of epitope mapping in Example 3, suggesting that the "footprint" of the antibody on Apple 3 overlaps with FIX-binding exosites in FXIa.

Example 5
Self-activation of FXI to FXIa on dextran sulfate The anti-FXI antibody of the invention in an 11-point dose titration starting at a concentration of 1 μM in a 3-fold dilution series.
In Corning 3575 unbound surface microplate, 50 mM HEPES, 1
50 mM NaCl, 5 mM CaCl ₂ , 0.1% PEG-8000 (pH 7.4)
Medium, Human FXI (Humanotropic Technologies, Inc., C.
Pre-incubated at 25 ° C. for 2 hours with at # HCXI-0150, final concentration 30 nM). Then, the self-activation reaction was started by the addition of dextran sulfuric acid (ACROS, Cat # 433240250, molecular weight of about 800 kDa, final concentration of 1 nM).
The reaction was allowed to proceed at 25 ° C. for 1 hour, at which time the Tecan Infinite M200
By continuously monitoring the fluorescence at 400/505 nm for 10 minutes using a plate reader, the newly activated FXIa enzyme activity was measured by the Z-GPR-AFC substrate (Sigma).
It was detected by the cutting rate of ma, Cat # C0980-10MG, final concentration 150 μM). The inhibition rate (%) for each data point is recalculated from the RFU / min data and GraphPa
d Prism software analyzed using a four-parameter equation of log (inhibitor) vs. response. The results are shown in Table 5.

Example 6
The ability of anti-FXI antibodies to block in vitro coagulation was evaluated using the activated partial thromboplastin time (aPTT) assay. Activated partial thromboplastin time (aPTT)
) Is a coagulation test that measures the activity of endogenous and common coagulation pathways.

Activated Partial Thromboplastin Time (aPTT) Assay The test is performed in sodium citrate plasma. Human plasma, blood from healthy donors of both sexes, Na tube of citrate (Sarthtedt coagulation 9NC / 10m)
Obtained by collecting in L). Blood is centrifuged at 1500 xg and plasma is collected.
The aPTT is examined for each donor, those within the normal range (28-40 seconds) are pooled, dispensed and stored at -80 ° C. Plasma from other species is commercially available (Innovative)
eResearch, Novi, MI). Test samples are prepared by adding (spiking) inhibitors or vehicles into plasma. Incubate these added samples (60 minutes at room temperature (RT)), followed by a coagulation analyzer (STA-R Evolut).
ion, Stago Diagnostics, Parsippany, NJ). Generally, the analyzer performs the following steps. Ellagic acid (Pacific Hemosta)
FXII is activated by the addition of sis, Thermo Fisher Scientific, Waltham, MA), and then the time to coagulation after calcium re-addition of the sample is measured. Inhibition of FXI prolongs the aPTT coagulation time. The results are shown in Table 6. The data are expressed as the rate of increase (%) with respect to the coagulation time of the vehicle control, 100% (2).
Concentrations that result in a 50% (1.5 ×) rate of increase in coagulation time are shown. aPT
The results of T are shown in FIGS. 6, 7, 8, 9 and 10.

Example 7
Surface plasmon resonance assay for evaluation of off-target binding of anti-FXI monoclonal antibodies to human and NHP coagulation cascade proteins Anti-factor FXI mAb αFXI-18611 IgG4 HC (S228P) (E1)
) (L105) / LC Kappa and αFXI-18623p IgG4 HC (S228)
P) (Q1) / LC Kappa, other human and NHP coagulation cascade proteins (Table 7)
A surface plasmon resonance (SPR) -based assay (Biacore T200) was used to determine potential non-specific interactions with. Anti-human IgG at about 500 RU to minimize potential background from co-purified Ig in plasma-derived proteins
The anti-FXI mAb was captured on a CM5 sensor chip immobilized using a (Fc) capture kit (GE Healthcare). A negative control antibody, the anti-respiratory syncytial virus (RSV) monoclonal antibody (mAb), was used as a reference and to assist in reducing background binding of plasma-derived proteins. Coupling kinetics were measured using 5 nM FXI analyte concentrations. All other coagulation cascade proteins are 500n
It was used at an analog concentration of M. Single concentration injection (n = 2) at 30 μL / min, 25 ° C., HB
It was carried out at S-EP + and pH 7.4.

Antifactors against human, cynomolgus monkey and rhesus monkey FXI and other human and NHP coagulation cascade proteins FXI mAb mAb αFXI-18611 IgG4
HC (S228P) (E1) (L105) / LC Kappa and αFXI-18623p
The kinetics of IgG4 HC (S228P) (Q1) / LC kappa binding was measured as described above. It is shown in FIGS. 11 and 12. Using the Biacore T200 evaluation software, the data were fitted to the 1: 1 binding model and the association rate constant ka (M- ¹ s).
^-1 ; where "M" is the molar concentration and "s" is the second) and the dissociation rate constant k _d (
s ^-1 ) was determined. The equilibrium dissociation constant KD (M) was calculated using these rate constants.

ΑFXI-18611 IgG4 HC (S228P) (E1) captured on the chip
(L105) / LC Kappa and αFXI-18623p IgG4 HC (S228P)
(Q1) / LC kappa did not show cross-reactivity to non-FXI coagulation cascade proteins (FIGS. 11 and 12). These monoclonal antibodies showed the expected levels of strong binding to human and cynomolgus monkey (and rhesus monkey) FXI proteins.

Example 8
Crab monkey femoral arteriovenous (AV) shunt thrombosis model αFXI-18623p IgG4 HC (S228P) (E1) / LC Kappa antibody antithrombotic efficacy, Merck, Sharp & Dohme Corp. Research
Laboratories, Kenilworth, NJ USA and Palo Al
Characterized in vivo in a cynomolgus monkey femoral arteriovenous (AV) shunt model developed at to, CA USA.

Study Design: These studies used an iterative method, in which each animal received two shunts over two consecutive study periods (see study outline in Figure 13). Each first
Non-antibody-containing vehicle (20 mM sodium acetate, 9) during the test period and the second test period
% Sucrose, pH 5.5) or αFXI-18623p IgG4 HC (S22)
8P) (E1) / LC kappa antibody (dose range 0.01-1.0 mg / kg) was administered to the monkeys. The difference in clot weight measured during the first (vehicle) test session and the second (antibody) test session determined the antithrombotic efficacy. That is, a greater reduction in clot weight during αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa antibody exposure compared to during vehicle exposure would indicate a greater antithrombotic effect. .. The use of the repeated pairing method allows an in-vivo evaluation of antithrombotic efficacy in animals.

Details of AV shunt placement operation: To carry out this model, femoral artery and venous catheters were attached to anesthetized cynomolgus monkeys. These catheters allowed the insertion and removal of AV shunts. The AV shunt consisted of a TYGON tube, which had a single silk suture that penetrated and hung across an opening in the tube.
Both arterial and venous catheters were closed to stop blood flow in order to place the AV shunt. An AV shunt was then placed between those two catheters. The timing of catheter placement and removal is shown in FIG. Once the shunt was in place, the catheter was opened and blood was allowed to flow through the shunt circuit in contact with the silk suture. The action of blood in contact with the suture promoted clot formation. The AV shunt was held in place for 40 minutes. A
To remove the V shunt, close both the arterial and venous catheters and A
The blood flow through the V shunt was stopped. The shunt was then removed and cut open to obtain silk sutures and blood clots. The blood clot was weighed. The data are shown as net clot weight, which is defined as total clot weight minus silk suture weight.

Activated partial thromboplastin time (aPTT) and prothrombin time (PT), which are coagulation biomarkers, and αFXI-18623p IgG4 HC (S228)
Circulating plasma levels of P) (E1) / LC kappa antibody were measured from blood samples collected through the experiments shown in FIG. Sta Compact Max Coagulation Analyzer (S)
Thaw (thawed from cynomolgus monkeys) using tago Graphicostic, Inc.
−80 ° C.) aPTT and PT were measured from citrated plasma. The Stago analyzer uses an electromagnetic mechanical clot detection system to measure the time of clot formation. For the aPTT assay, 50 microliters of plasma with 50 μL of ellagic acid mixture (APTT-XL)
, Pacific Hemostasis; Fisher Scientifics c
At # 10-0402) was mixed at 37 ° C. for 3 minutes. 50 μl 0.025M Calcium Chloride (Sta-CaCl ₂ 0.025M, Stago Graphicostic, In)
c. , Cat # 00367) was added to the mixture, and the time to clot formation was measured. For the PT assay, 50 microliters of plasma were incubated at 37 ° C. for 4 minutes. 1
00 μL thromboplastin reagent (Neoplastine Cl Plus 10,
Stago Digital, Inc. , Cat # 0267) to start the timing of blood clot formation. Plasma was measured as follows.
Antibodies in cynomolgus monkey plasma were quantified using a common hIgG4 immunoassay based on electrochemical luminescence. Biotinylated goat anti-human IgG (H + L) from Bethyl (cat # A80-319B) as a capture reagent, and Southern Bio for detection reagents
SulfoTAG-labeled mouse anti-human IgG (Fc) from tech (cat # 9190-01)
Specific) was used to establish the assay. The assay was certified and the lower limit of quantification for the assay was determined to be 40 ng / mL with a minimum required dilution of 100.

14A-D show thrombus formation (FIGS. 14A, 14B), aPTT (FIG. 14C) and PT.
ΑFXI-18623p IgG4 HC (S228P) (E1) relative to (FIG. 14D)
/ Summarizes the effects of administration of LC kappa antibody. Table 8 shows αFXI-18623p IgG4 HC (S228P) (E) for blood clot weight in the cynomolgus monkey AV shunt model.
1) / Summarizes the effects of LC kappa antibody. Table 9 shows αFXI-18623p IgG4 HC (S228) for aPTT and PT in the cynomolgus monkey AV shunt model.
P) The effects of the (E1) / LC kappa antibody are summarized.

ΑFXI-18623p IgG, as shown in FIGS. 14A, 14B and Table 8.
4 HC (S228P) (E1) / LC kappa antibody showed dose-dependent and plasma concentration-dependent reduction in clot weight, with full efficacy (90-100% clot reduction) of 1 μg / mL.
It was observed in plasma [antibodies] above (about 10 nM). As shown in FIG. 14C and Table 9, the antibody showed a dose-dependent and plasma concentration-dependent increase in aPTT. Plasma concentration of 2.4 μg / mL (~ 17 nM) αFXI-18623p IgG4 H
The C (S228P) (E1) / LC kappa antibody resulted in a 93% increase in aPTT, while 29 μg / mL (~ 200 nM) (highest dose tested) αFXI-18623.
p IgG4 HC (S228P) (E1) / LC kappa antibody is 143 in aPTT
Brought an increase of%. As shown in FIG. 14D and Table 9, unlike aPTT, PT varied by less than 10% over the assessed concentration of the antibody, which is consistent with the selective effect of FXI inhibition on the endogenous coagulation pathway. I was doing it.

Example 9
Cynomolgus monkey template Bleeding time model Merck, Sharp & Dohme Corp. Research Labor
atories, Kenilworh, NJ USA and Palo Alto, CA
Anti-FXI in the cynomolgus monkey template bleeding time model developed in USA
The bleeding tendency of mAb αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa was characterized in vivo. This model has already been used to show a significant increase in template bleeding time for multiple anatomical sites in triple antiplatelet therapy (Cai et al., Eur J Pharmacol 758: 107-114).
(2015)).

To carry out this model, a spring-loaded lancet was used to induce bleeding in the buccal mucosa (inner lip), finger toes and distal tail at various time points to determine template bleeding time.

Bleeding time test: A bleeding time test was performed on anesthetized cynomolgus monkeys as follows.

Each test area (buccal mucosa, finger toes or distal tail) was examined to identify the appropriate incision site for bleeding induction.

-To induce bleeding, a spring-loaded lancet was placed tightly at the selected test site and actuated to produce a uniform linear incision. The lancet specifications determined the incision dimensions.

-Blood from the incision site was allowed to flow freely and monitored until bleeding stopped continuously for 30 seconds. This defined the bleeding time (BT). BT was recorded for each BT site. During the BT measurement, the distal tail incision site was perfused with warm sterile Ringer's lactate solution and the finger toe site was immersed in warm sterile Ringer's lactate solution. The application of lactated Ringer's solution improved the ability to observe blood flow at these sites.

Study Design: Each study consisted of three 30-minute template bleeding time studies (BT) in three study areas (see study outline in Figure 15). The first BT determined baseline bleeding. The second BT was a compound-free vehicle (20).
3 minute IV injection (4.17) of mM sodium acetate, 9% sucrose, pH 5.5)
It was carried out 70 minutes after (ml / kg) (treatment 1). The third BT was a compound-free vehicle or αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa (1).
It was performed 70 minutes after IV infusion (4.17 ml / kg) (treatment 2) for 3 minutes (0 mg / kg). Bleeding was monitored and bleeding time was recorded as described above. The time at which bleeding stopped was recorded for each site. Blood samples are taken on a regular basis and αFXI-18623p Ig
Circulating plasma levels of G4 HC (S228P) (E1) / LC kappa, aPTT and PT
It was determined.

Each test animal was submitted to two study sessions. In study session 1, the vehicle and subsequent vehicles constituted process 1 and process 2, respectively. In Study Session 2, the vehicle followed by 10 mg / kg IV αFXI-1862
3p IgG4 HC (S228P) (E1) / LC kappa constituted treatment 1 and treatment 2, respectively.

The 70 minutes between the end of test substance infusion and the start of bleeding time assessment represented the timing in the AV shunt model for thrombus mass measurement (shunt placement 30 minutes after treatment +
40 minutes of blood flow through the shunt). Based on the PK / PD primate modeling studies already described, the test dose of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa 10 mg / kg IV is αFXI-18623p IgG4 HC (
It was estimated to achieve 10 times _{the expected human C max} of S228P) (E1) / LC kappa.

Activated partial thromboplastin time (aPTT) and prothrombin time (PT), which are coagulation biomarkers, and αFXI-18623p IgG4 HC (S228)
Circulating plasma levels of P) (E1) / LC kappa were measured from blood samples collected through the experiments shown in FIG. Sta-R Evolution solidification analyzer (Sta)
APTT and PT were measured from thawed (-80 ° C) citrated plasma collected from animals using go Dynamic, Inc.). The coagulation analyzer uses an electromagnetic mechanical clot detection system to measure the time to clot formation. For the aPTT assay, the analyzer combines 50 μL of plasma with 50 μL of ellagic acid (APTT-XL, Pacific H).
emostasis; Fisher Scientifics cat # 10-0402
) And in a cuvette, then incubate at 37 ° C. for 3 minutes. Then 5
0 μL of 0.025M calcium chloride (Sta-CaCl ₂ 0.025M, Tag)
o Digital, Inc. , Cat # 00367) is added to the mixture to initiate coagulation and the time to clot formation is measured. For the PT assay, 50 μL of plasma was incubated in a cuvette at 37 ° C. for 4 minutes. 100 μL of solubilized thromboplastin reagent (Triniclot PT Excel, TCog, Inc., cat #
Blood clot formation was initiated by the addition of T1106).

ΑFXI-18623p IgG4 HC (S228P) (E1) / LC kappa in rhesus plasma was quantified using a common hIgG4 immunoassay based on electrochemical luminescence. Biotinylated goat anti-human IgG (H + L) from Bethyl (cat # A80-319B) as a capture reagent, and Southern Biotech (c) for a detection reagent.
The assay was established using sulfoTAG-labeled mouse anti-huIgG (Fc-specific) from at # 9190-01). This assay has been certified and the lower limit of quantification for this assay is 10.
It was determined to be 41 ng / mL with a minimum required dilution of 0.

16A-16F show the buccal mucosa (FIGS. 16A, 16D) and finger toes (FIGS. 16B, 16E).
Vehicles and 10 mg / kg IV αFXI-18623p IgG4 in 6 cynomolgus monkeys for template bleeding time and distal tail (Fig. 16C, Fig. 16F)
The effects of administration of HC (S228P) (E1) / LC kappa are summarized. Vehicle-vehicle as process 1 and process 2 in research session 1 and research session 2
Vehicle as treatments 1 and 2 in-αFXI-18623p IgG4 HC
For (S228P) (E1) / LC kappa, the effect on bleeding time was evaluated by comparing the absolute bleeding time (left panel) and the rate of change (%) (right panel) in bleeding time. Vehicle vs. αFXI-18623p IgG4 HC (S228P) (E1) /
Absolute bleeding time of LC kappa and vehicle-vehicle vs. vehicle-αFXI-1862
A comparison of both the rate of change in bleeding time of 3p IgG4 HC (S228P) (E1) / LC kappa is a comparison of αFXI-18623p IgG4 HC (S228P) at this test dose.
) (E1) / LC kappa administration did not detect a statistically significant change in bleeding time at any of the test sites.

ΑFXI obtained at 10 mg / kg IV test dose in cynomolgus monkey bleeding time test
-18623p IgG4 HC (S228P) (E1) / LC kappa plasma concentration is 29
It was 0.7 ± 17.2 (mean ± SEM) μg / mL (~ 1938.2 nM). Plasma a
The PTT value is 31.0 ± 0.5 seconds at baseline, while 10 mg / kg
It was 71.3 ± 1.6 seconds (2.3-fold increase) after administration of αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa of IV. Plasma PT values were 12.7 ± 0.1 seconds at baseline, while 10 mg / kg IV αFXI-186
12.8 ± 0 after administration of 23p IgG4 HC (S228P) (E1) / LC kappa
.. It was 24 seconds (no obvious increase was observed).

Example 10
ΑFXI-18623p IgG4 H after multiple intravenous doses in rhesus monkeys
Evaluation of C (S228P) (E1) / LC Kappa Pharmacokinetics (PK) and Pharmacodynamics (PD) αFXI-18623p IgG4 HC (S228P) (E1) / LC Kappa PK
PD properties were characterized in vivo in rhesus monkeys. The purpose was to assess PK properties and establish a PK / PD relationship after a total of two weekly doses.

Study Design: Non-Compound Vehicle (10 mM Sodium Acetate (pH 5.5), 7% Sucrose, 0.02% PS-80) or αFXI-18623p IgG4 HC (S)
228P) (E1) / LC kappa was administered (IV) to rhesus monkeys (4 animals per dose group) at 5 dose levels of 0.1, 0.3, 1, 3 and 6 mg / kg. The duration of this study was 22 days and 1.5 mL of blood was taken to determine drug levels and activated partial thromboplastin time (aPTT).

As shown in Table 10, coagulation biomarkers (aPTT) and αFXI-18623p IgG4 HC (S228P) (E) from blood samples collected throughout the experiment.
1) / The circulating plasma level of LC kappa was measured.

Sta-R Evolution Coagulation Analyzer (Stago Graphicostic,
APTT from thawed (-80 ° C) citrated plasma collected from animals using Inc)
Was measured. The coagulation analyzer uses an electromagnetic mechanical clot detection system to measure the time to clot formation. For the aPTT assay, the analyzer combines 50 μL of plasma with 50 μL of ellagic acid (APTT-XL, Pacific Hemostasis; Fisher Di).
Mix with agnostics cat # 10-0402) in a cuvette, then incubate at 37 ° C. for 3 minutes. Then 50 μL of 0.025 M calcium chloride (
Sta-CaCl ₂ 0.025M, Stago Diagnostic, Inc. , C
At # 00367) is added to the mixture to initiate coagulation and the time to clot formation is measured.

ΑFXI-18623p IgG4 HC (S228P) (E1) / LC kappa in rhesus plasma was quantified using a common hIgG4 immunoassay based on electrochemical luminescence. Biotinylated goat anti-huIgG (H + L) from Bethyl (cat # A80-319B) as a capture reagent, and Southern Biotech (c) for a detection reagent.
The assay was established using sulfoTAG-labeled mouse anti-huIgG (Fc-specific) from at # 9190-01). This assay has been certified and the lower limit of quantification for this assay is 10.
It was determined to be 41 ng / mL with a minimum required dilution of 0.

Non-compartment (NCA) method (Gabrielsson and Weiner, 20)
00) using αFXI-18623p IgG4 HC (S228P) (E1) / L
Individual animal plasma concentration-time data for C-kappa were analyzed. Phoenix 32
WinNonlin 6.3 (version 6.3.0.395, Certar LP)
.. St. All PK parameters were estimated or calculated using Louis, MO, 2012). Model 201 (IV) was used for non-compartment analysis. All concentration data and PK parameters were rounded to 3 significant figures. Less than the lower limit of quantification (<LLO
Samples with a concentration value of Q) were excluded from PK analysis and mean data calculation. For the purpose of graphing, values below LLOQ were set to 1/2 of the minimum reportable concentration for individual animal concentration-time plots.

GraphPad Prism version 7.00 (GraphPad Softwa)
using re Inc) to characterize the relationship between exposure and aPTT, S-shaped _{Ema
The x-} response (PK / PD) model was used. In this model, the E _max value corresponds to the maximum increase in aPTT obtained from the baseline, and the EC ₅₀ value corresponds to the half effect concentration. Variability, 95% confidence intervals for _{The EC 50} values obtained by the software (CI)
Shown as.

Results: Individual concentration-time profiles for αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa are shown in FIG. 17A. Non-linearity was observed for all PK parameters. The average clearance value is the lowest dose tested (0.1 mg / k)
From about 8 mL / kg / day for g) to about 4 for the highest dose tested (6 mg / kg)
It decreased to mL / kg / day. The concentration-time profile of aPTT is shown in FIG. 17B. a
A dose-dependent increase in PTT was observed. The relationship between αFXI-18623p IgG4 HC (S228P) (E1) / LC kappa plasma concentration and aPTT, which is best represented by the S-shaped E _{max model, adequately indicated this relationship.} αFXI-18623p IgG4
HC (S228P) (E1) / LC estimation _{The EC 50} values relating kappa of about 3.6 g / mL
Met.

Although the present invention is described herein with respect to exemplary embodiments, it should be understood that the invention is not limited thereto. Those skilled in the art and those utilizing the teachings will recognize additional modifications and embodiments within the scope of the invention. Therefore, the present invention is limited only by the scope of claims herein.

Claims (48)

(I) Contains at least 6 complementarity determining regions (CDRs) of anti-FXI antibodies of the αFXI-18623p family, αFXI-18611p family or αFXI-18611 family, or (ii) one or more of the 6 CDRs is 1 The αFXI-18623p family, with 2, or 3 amino acid substitutions, additions, deletions or combinations thereof.
An antibody or antigen-binding fragment comprising at least six complementarity determining regions (CDRs) of an anti-FXI antibody of the αFXI-18611p family or the αFXI-18611 family, wherein.
Antibodies of the αFXI-18623 family include a heavy chain (HC) variable region having the amino acid sequence set forth in SEQ ID NO: 28 or 29 and an LC variable region having the amino acid sequence set forth in SEQ ID NO: 30.
Antibodies of the αFXI-18611p family include an HC variable region having the amino acid sequence set forth in SEQ ID NO: 21 or 22 and a light chain (LC) variable region having the amino acid sequence set forth in SEQ ID NO: 25.
Antibodies of the αFXI-18611 family include antibody or antigen binding comprising an HC variable region having the amino acid sequence set forth in SEQ ID NO: 23 or 24 and an LC variable region having the amino acid sequence set forth in SEQ ID NO: 25. Sex fragment. Six CDRs are the HC CDR1, CDR2 and CDR3 of the anti-FXI antibody of the FXI-18623p family, αFXI-18611p family or αFXI-18611 family, and the anti-FXI of the FXI-18623p family, αFXI-18611p family or αFXI-18611 family. The antibody or antigen-binding fragment of claim 1, comprising the LC of the antibody, CDR1, CDR2 and CDR3. The antibody or antigen-binding fragment has CDR1, CDR2 and CDR3 of the HC variable domain having the amino acid sequence set forth in SEQ ID NO: 28 or 29, and CDR1 of the LC variable domain having the amino acid sequence set forth in SEQ ID NO: 30. , CDR2 and CDR3; or CDR1, CDR2 and CDR3 of the HC variable domain having the amino acid sequence set forth in SEQ ID NO: 21, 22, 23 or 24, and SEQ ID NO: 25.
CDR1, CDR2 and CD of LC variable domains having the amino acid sequence shown in
The antibody or antigen-binding fragment according to claim 2, which comprises R3. HC in which the antibody comprises the amino acid sequence set forth in SEQ ID NO: 16, 17, 18 or 19.
The antibody or antigen-binding fragment according to any one of claims 1 to 3, which comprises a constant domain. The antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody comprises an LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments
(A) Heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 28 and light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 30.
(B) A heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 29 and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 30.
(C) A heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 21 and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25.
(D) A heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 22 and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25.
(E) A heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 23 and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25.
(F) A heavy chain (HC) variable domain having the amino acid sequence shown in SEQ ID NO: 24 and a light chain (LC) variable domain having the amino acid sequence shown in SEQ ID NO: 25.
(G) HC variable domain framework is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
Includes amino acid substitutions, additions, deletions or combinations thereof, (a), (b), (c),
Variants of (d), (e) or (f), or (h) LC variable region frameworks are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
Includes amino acid substitutions, additions, deletions or combinations thereof, (a), (b), (c),
The antibody or antigen-binding fragment according to claim 1 or 3, which comprises a variant of (d), (e), (f) or (g). The antibody further comprises an HC containing the amino acid sequence set forth in SEQ ID NOs: 16, 17, 18, 19
The antibody or antigen-binding fragment according to claim 6, which comprises a constant domain. The antibody or antigen-binding fragment of claim 6, wherein the antibody further comprises an LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20. An antibody or antigen-binding fragment binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits FXI activation and / or factor XIa-mediated activation of factor IX.
The antibody or antigen-binding fragment according to claim 1, 2, 3, 4, 5, 6, 7 or 8. The antibody
(A) Heavy chain complementarity determining regions (HC-C) having the amino acid sequence shown in SEQ ID NO: 1.
DR) 1, HC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, HC having variable and constant domains including HC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 or 4, and HC. (B) Light chain complementarity determining regions (LC-C) having the amino acid sequence shown in SEQ ID NO: 5.
DR) 1, LC having a variable domain including LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 6 and LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 7 and LC having a constant domain.
The antibody or antigen-binding fragment according to claim 1 or 3. The antibody or antigen-binding fragment of claim 10, wherein the antibody further comprises an HC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16, 17, 18 or 19. The antibody or antigen-binding fragment of claim 10, wherein the antibody further comprises an LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20. The antibody
(A) Heavy chain complementarity determining regions (HC-C) having the amino acid sequence shown in SEQ ID NO: 8.
DR) 1. It has a variable domain and a constant domain constituting a heavy chain containing HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 9 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 10. HC and (b) light chain complementarity determining regions having the amino acid sequence set forth in SEQ ID NO: 11 (LC-
CDR) 1, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 12 and LC having a variable domain and a constant domain including LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 13.
The antibody or antigen-binding fragment according to claim 1 or 3. HC in which the antibody comprises the amino acid sequence set forth in SEQ ID NO: 16, 17, 18 or 19.
The antibody or antigen-binding fragment according to claim 13, which comprises a constant domain. 13. The antibody or antigen-binding fragment of claim 13, wherein the antibody comprises an LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20. The antibody
SEQ ID NOs: 33, 35, 37, 39, 45, 47, 49, 51, 57, 59, 61, 63
, 69, 71, 73 or HC having the amino acid sequence shown in 75, and 1, 2
An LC having the amino acid sequence shown in SEQ ID NO: 26, as well as its variants, including 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. And 1, 2, 3, 4, 5,
Includes variants thereof containing 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is the Apple 3 domain of coagulation factor XI (FXI). The antibody or antigen-binding fragment according to claim 1 or 3, which binds to and / or inhibits factor XIa-mediated activation of factor IX. The antibody
HC having the amino acid sequence set forth in SEQ ID NO: 41, 43, 53, 55, 65, 67, 77 or 79, and 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Its variants, including amino acid substitutions, additions, deletions or combinations thereof, and LCs having the amino acid sequence set forth in SEQ ID NO: 31, and 1, 2, 3, 4, 5,
Includes variants thereof containing 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, wherein the antibody or antigen binding fragment is the Apple 3 domain of coagulation factor XI (FXI). The antibody or antigen-binding fragment according to claim 1 or 3, which binds to and / or inhibits factor XIa-mediated activation of factor IX. (A) (i) The amino acid sequence shown in heavy chain complementarity determining regions (HC-CDR) 1, SEQ ID NO: 9 having the heavy chain (HC) framework and the amino acid sequence shown in SEQ ID NO: 8. HC-CDR2 having and H having the amino acid sequence shown in SEQ ID NO: 10
C-CDR3;
(Ii) HC framework and heavy chain complementarity determining regions (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: HC-CDRs having the amino acid sequence shown in 3.
3;
(Iii) HC framework and heavy chain complementarity determining regions (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: HC-CDs having the amino acid sequence shown in 4.
R3;
(Iv) At least one of HC CDR1, HC-CDR2 or CDR3 is 1,
(I), (ii), comprising 2 or 3 amino acid substitutions, additions, deletions or combinations thereof.
) Or (iii) variants; or (v) the HC framework has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. Including, (i), (ii), (iii)
Or a heavy chain (HC) having a variable domain containing a variant of (iv) and a constant domain;
(B) (i) Light chain complementarity determining regions (LC-CDR) 1 having the amino acid sequence shown in the light chain (LC) framework and SEQ ID NO: 11, the amino acid sequence shown in SEQ ID NO: 12. LC-CDR2 and LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 13;
(Ii) LC framework and light chain complementarity determining regions (LC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 5, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 6 and SEQ ID NO: LC-CDRs having the amino acid sequence shown in 7.
3;
(Iii) At least one of LC CDR1, LC-CDR2 or LC-CDR3
One contains one, two or three amino acid substitutions, additions, deletions or combinations thereof, (i).
Or a variant of (ii); or (iv) the LC framework comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof. , (I), (ii) or (i)
A light chain (LC) having a variable domain containing a variant of ii) and a constant domain; or HC from (c) (a) and LC from (b).
An antibody comprising, wherein the antibody binds to the Apple 3 domain of coagulation factor XI (FXI) and inhibits the activation of FXI and / or the factor XIa-mediated activation of factor IX. The antibody according to claim 18, wherein the HC constant domain comprises the amino acid sequence set forth in SEQ ID NO: 16, 17, 18 or 19. The antibody according to claim 18 or 19, wherein the LC constant domain comprises the amino acid sequence set forth in SEQ ID NO: 20. An isolated nucleic acid molecule encoding a light chain variable domain or a heavy chain variable domain of any one of the antibodies or antigen-binding fragments according to any one of claims 1-20. A composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier or diluent. A method for treating a thromboembolic disorder or disease in a subject, which comprises administering to the subject an effective amount of the antibody or antigen-binding fragment according to any one of claims 1 to 20. Use of the antibody according to any one of claims 1 to 20, for the manufacture of a medicament for treating a thromboembolic disorder or disease. The antibody according to any one of claims 1 to 20, for the treatment of a thromboembolic disorder or disease. A human antibody or antigen-binding fragment that binds to an epitope on coagulation factor XI (FXI), wherein the epitope is the amino acid sequence DIFFNTVF (SEQ ID NO:) determined by use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). 82) and amino acid sequence PSDR
A human antibody or antigen-binding fragment comprising IKKSKALSG (SEQ ID NO: 83). 26. The human antibody of claim 26, wherein the antibody comprises (i) a human IgG1 constant domain or a variant or modified derivative thereof, or (ii) a human IgG4 constant domain or a variant or modified variant thereof. 26. The antibody comprises an IgG4 constant domain comprising substitution of a serine residue at position 228 (EU numbering) or position 108 (as shown herein) with a proline residue.
The human antibody described. SEQ ID NOs: 33, 35, 37, 39, 45, 47, 49, 51, 57, 59, 61, 63
, 69, 71, 73 or 75 with the heavy chain having the amino acid sequence and SEQ ID NO: 2.
Antibodies containing light chains having the amino acid sequence shown in 6 or SEQ ID NOs: 41, 43, 5
Cross-blocking or binding an antibody comprising a heavy chain having the amino acid sequence set forth in 3, 55, 65, 67, 77 or 79 and a light chain having the amino acid sequence set forth in SEQ ID NO: 31. An antibody or antigen-binding fragment that competes with. 29. The human antibody of claim 29, wherein the antibody comprises (i) a human IgG1 constant domain or a variant or modified derivative thereof, or (ii) a human IgG4 constant domain or a variant or modified variant thereof. 30. The antibody comprises an IgG4 constant domain comprising substitution of a serine residue at position 228 (EU numbering) or position 108 (as shown herein) with a proline residue.
The human antibody described. (I) Heavy chain complementarity determining regions (HC-C) having the amino acid sequence shown in SEQ ID NO: 1.
DR) 1, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 2, HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 3 or 4, or SEQ ID NO: 8
A heavy chain variable domain comprising HC-CDR1 having the amino acid sequence shown in, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 9 and HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 10. , And (ii) light chain complementarity determining regions (LC-) having the amino acid sequence set forth in SEQ ID NO: 5.
CDR) 1, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 6, LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 7, or LC having the amino acid sequence shown in SEQ ID NO: 11. -CDR1, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 12 and LC having the amino acid sequence shown in SEQ ID NO: 13.
-A method for producing an antibody or antigen-binding fragment containing a light chain variable domain containing CDR3, wherein a host cell containing a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain is prepared. A production method comprising culturing the host cell under conditions sufficient to produce an antibody or antigen-binding fragment and for a time sufficient to produce it. 32. The production method according to claim 32, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21, 22, 23 or 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25. 32. The production method of claim 32, wherein the antibody comprises a heavy chain constant domain of an IgG1, IgG2, IgG3 or IgG4 isotype. 32. The production method of claim 32, wherein the antibody comprises a heavy chain constant domain of IgG4 isotype. 32. The production method of claim 32, wherein the antibody comprises a heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16, 17, 18 or 19. 32. The production method according to claim 32, wherein the light chain comprises a human kappa light chain or a human lambda light chain. 32. The production method of claim 32, wherein the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20. The production method according to claim 32, wherein the host cell is a Chinese hamster ovary cell or a human fetal kidney 293 cell. 32. The production method according to claim 32, wherein the host cell is a yeast or filamentous fungal cell. The composition according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment is obtained from a host containing a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain. 41. The composition of claim 41, wherein the antibody comprises a heavy chain constant domain of an IgG1, IgG2, IgG3 or IgG4 isotype. 41. The composition of claim 41, wherein the antibody comprises a heavy chain constant domain of IgG4 isotype. 41. The composition of claim 41, wherein the antibody comprises a heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 16, 17, 18 or 19. 41. The composition of claim 41, wherein the light chain comprises a human kappa light chain or a human lambda light chain. 41. The composition of claim 41, wherein the antibody comprises a light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 20. 41. The composition of claim 41, wherein the host cell is a Chinese hamster ovary cell or a human fetal kidney 293 cell. 41. The composition of claim 41, wherein the host cell is a yeast or filamentous fungal cell.

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